Protease variants and compositions

ABSTRACT

The present invention relates to protease subtilase enzyme, characterized by an insertion in at least one active site loop. The enzymes exhibit improved wash performance in a detergent in comparison to its parent enzyme if it is a subtilase variant.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of Ser. No. 09/196,281 filedNov. 19, 1998, now allowed, and claims priority under 35 U.S.C. 119 ofDanish application no 1332/97 filed Nov. 21, 1997, the contents of whichare fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to novel mutant protease enzymes or enzymevariants, comprising insertions in one or more active site loops, usefulin formulating detergent compositions and exhibiting improved washperformance in detergents; cleaning and detergent compositionscontaining said enzymes; mutated genes coding for the expression of saidenzymes when inserted into a suitable host cell or organism; and suchhost cells transformed therewith and capable of expressing said enzymevariants.

[0004] 2. Description of Related Art

[0005] In the detergent industry enzymes have for more than 30 yearsbeen implemented in washing formulations. Enzymes used in suchformulations comprise proteases, lipases, amylases, cellulases, as wellas other enzymes, or mixtures thereof. Commercially most importantenzymes are proteases.

[0006] An increasing number of commercially used proteases are proteinengineered variants of naturally occurring wild type proteases, e.g.DURAZYM® (Novo Nordisk A/S), RELASE® (Novo Nordisk A/S), MAXAPEM®(Gist-Brocades N.V.), PURAFECT® (Genencor International, Inc.).

[0007] Further a number of protease variants are described in the art,such as in EP 130756 (GENENTECH)(corresponding to U.S. Reissue Pat. No.34,606 (GENENCOR)); EP 214435 (HENKEL); WO 87/04461 (AMGEN); WO 87/05050(GENEX); EP 260105 (GENENCOR); Thomas, Russell, and Fersht (1985) Nature318 375-376; Thomas, Russell, and Fersht (1987) J. Mol. Biol. 193803-813; Russel and Fersht Nature 328 496-500 (1987); WO 88/08628(Genex); WO 88/08033 (Amgen); WO 95/27049 (SOLVAY S.A.); WO 95/30011(PROCTER & GAMBLE COMPANY); WO 95/30010 (PROCTER & GAMBLE COMPANY); WO95/29979 (PROCTER & GAMBLE COMPANY); US 5.543.302 (SOLVAY S.A.); EP 251446 (GENENCOR); WO 89/06279 (NOVO NORDISK A/S); WO 91/00345 (NOVONORDISK A/S); EP 525 610 Al (SOLVAY); and WO 94/02618 (GIST-BROCADESN.V.).

[0008] However, even though a number of useful protease variants havebeen described, there is still a need for new improved proteases orprotease variants for a number of industrial uses.

[0009] Therefore, an object of the present invention is to provideimproved proteases or protein engineered protease variants, especiallyfor use in the detergent industry.

SUMMARY OF THE INVENTION

[0010] The present inventors have identified that it is possible toconstruct variants of BLSAVI (Savinase®), having improved washperformance in detergent, as compared to the parent wildtype BLSAVI, byintroducing at least one insertion in at least one of the active siteloops in said BLSAVI.

[0011] It is predicted that it will be possible to make similar variantsin other subtilases, which are similar to BLSAVI.

[0012] Further it is predicted that it is possible to isolate fromnature and identify naturally occurring parent or wildtype subtilases,having improved wash performance in a detergent, as compared to BLSAVI,by specifically screening for such parent wildtype subtilases comprisingat least one active site loop, which is longer than the correspondingactive site loop in BLSAVI.

[0013] Accordingly, in a first aspect the invention relates to anisolated subtilase enzyme, having improved wash performance in adetergent, as compared to BLSAVI, having an amino acid sequence which isat least 40% identical to the amino acid sequence of the mature BLSAVI,and characterized by that at least one of the active site loops, in saidisolated subtilase, is longer than the corresponding active site loop inBLSAVI, whereby such active site loops regions, in said isolatedsubtilase, is having the minimum amino acid length as specified from thegroup below comprising:

[0014] (a) the region (both of the end amino acids included) betweenamino acid residue from 33 to 43 is at least 11 amino acid long (i.e. atleast one amino acid insertion, as compared to BLSAVI);

[0015] (b) the region (both of the end amino acids included) betweenamino acid residue 95 to 103 is at least 9 amino acids long (i.e. atleast one amino acid insertion, as compared to BLSAVI);

[0016] (c) the region (both of the end amino acids included) betweenamino acid residue 125 to 132 is at least 8 amino acids long (i.e. atleast one amino acid insertion, as compared to BLSAVI);

[0017] (d) the region (both of the end amino acids included) betweenamino acid residue 153 to 173 is at least 21 amino acids long (i.e. atleast one amino acid insertion, as compared to BLSAVI);

[0018] (e) the region (both of the end amino acids included) betweenamino acid residue 181 to 195 is at least 15 amino acids long (i.e. atleast one amino acid insertion, as compared to BLSAVI);

[0019] (f) the region (both of the end amino acids included) betweenamino acid residue 202 to 204 is at least 3 amino acids long (i.e. atleast one amino acid insertion, as compared to BLSAVI); and

[0020] (g) the region (both of the end amino acids included) betweenamino acid residue 218 to 219 is at least 3 amino acids long (i.e. atleast one amino acid insertion, as compared to BLSAVI).

[0021] In a second aspect the invention relates to an isolated DNAsequence encoding a subtilase variant of the invention. In a thirdaspect the invention relates to an expression vector comprising anisolated DNA sequence encoding a subtilase variant of the invention.

[0022] In a fourth aspect the invention relates to a microbial host celltransformed with an expression vector according to the fourth aspect.

[0023] In a further aspect the invention relates to the production ofthe subtilisin enzymes of the invention by inserting an expressionvector according to the fourth aspect into a suitable microbial host,cultivating the host to express the desired subtilase enzyme, andrecovering the enzyme product.

[0024] Further the invention relates to a composition comprising asubtilase variant of the invention.

[0025] Even further the invention relates to the use of the mutantenzymes for a number of industrial relevant uses, in particular for usein cleaning compositions and cleaning compositions comprising the mutantenzymes, especially detergent compositions comprising the mutantsubtilisin enzymes.

[0026] Definitions

[0027] Prior to discussing this invention in further detail, thefollowing term will first be defined. NOMENCLATURE OF AMINO ACIDS A =Ala = Alanine V = Val = Valine L = Leu = Leucine I = Ile = Isoleucine P= Pro = Proline F = Phe = Phenylalanine W = Trp = Tryptophan M = Met =Methionine G = Gly = Glycine S = Ser = Serine T = Thr = Threonine C =Cys = Cysteine Y = Tyr = Tyrosine N = Asn = Asparagine Q = Gln =Glutamine D = Asp = Aspartic Acid E = Glu = Glutamic Acid K = Lys =Lysine R = Arg = Arginine H = His = Histidine X = Xaa = Any amino acidNOMENCLATURE OF NUCLEIC ACIDS A = Adenine G = Guanine C = Cytosine T =Thymine (only in DNA) U = Uracil (only in RNA)

[0028] Nomenclature of Variants

[0029] In describing the various enzyme variants produced orcontemplated according to the invention, the following nomenclatureshave been adapted for ease of reference:

[0030] Original Amino Acid(s)—Position(s)—Substituted Amino Acid(s)

[0031] In the case when the original amino acid residue may be any aminoacid residue, a short hand notation may at times be used indicating onlythe position and substituted amino acid,

[0032] Position—Substituted Amino Acid

[0033] Such a notation is particular relevant in connection withmodification(s) in homologous subtilases (vide infra).

[0034] Similarly when the identity of the substituting amino acidresidue(s) is immaterial,

[0035] Original Amino Acid—Position

[0036] When both the original amino acid(s) and substituted aminoacid(s) may comprise any amino acid, then only the position(s) isindicated, e.g., 170.

[0037] When the original amino acid(s) and/or substituted amino acid(s)may comprise more than one, but not all amino acid(s), then the selectedamino acids are indicated inside brackets { }.

[0038] Original Amino Acid—Position—{Substituted Amino Acid₁, . . . ,Substituted Amino Acid_(n)}

[0039] For specific variants the specific three or one letter codes areused, including the codes Xaa and X to indicate any amino acid residue.

[0040] Substitutions:

[0041] The substitution of glutamic acid for glycine in position 195 isdesignated as:

[0042] Gly195Glu or G195E

[0043] or the substitution of any amino acid residue acid for glycine inposition 195 is designated as:

[0044] Glu195Xaa or G195X or

[0045] Glu195 or G195

[0046] The substitution of serine for any amino acid residue in position170 would thus be designated

[0047] Xaa170Ser or X170S. or

[0048] 170Ser or 170S

[0049] Such a notation is particular relevant in connection withmodification(s) in homologous subtilases (vide infra). 170Ser is thusmeant to comprise e.g. both a Lys170Ser modification in BASBPN andArg170Ser modification in BLSAVI. See FIG. 1 in relation to theseexamples.

[0050] For a modification where the original amino acid(s) and/orsubstituted amino acid(s) may comprise more than one, but not all aminoacid(s), the substitution of glycine, alanine, serine or threonine forarginine in position 170 would be indicated by

[0051] Arg1 70{Gly,Ala,Ser,Thr} or R170{G,A,S,T}

[0052] to indicate the variants

[0053] R170G, R170A, R170S, and R170T.

[0054] Deletions:

[0055] A deletion of glycine in position 195 will be indicated by:

[0056] Gly195* or G195*

[0057] Correspondingly the deletion of more than one amino acid residue,such as the deletion of glycine and leucine in positions 195 and 196will be designated

[0058] Gly195*+Leu196* or G195*+L196*

[0059] Insertions:

[0060] The insertion of an additional amino acid residue such as e.g. alysine after G195 is:

[0061] Gly195GlyLys or G195GK; or

[0062] when more than one amino acid residue is inserted, such as e.g. aLys, Ala and Ser after G195 this is:

[0063] Gly195GlyLysAlaSer or G195GKAS (SEQ ID NO: 1)

[0064] In such cases the inserted amino acid residue(s) are numbered bythe addition of lower case letters to the position number of the aminoacid residue preceding the inserted amino acid residue(s). In the aboveexample the sequences 194 to 196 would thus be: 194 195 196 BLSAVI A -G - L (SEQ ID NO: 2) 194 195 195a 195b 195c 196 VariantA - G - K -  A -  S -  L

[0065] In cases where an amino acid residue identical to the existingamino acid residue is inserted it is clear that a kind of degeneracy inthe nomenclature arises. If for example a glycine is inserted after theglycine in the above example this would be indicated by G195GG. The sameactual change could just as well be indicated as Al 94AG for the changefrom 194 195 196 BLSAVI to A - G - L (SEQ ID NO: 3) 194 195 195a 196Variant A - G - G -  L 194 194a 195 196

[0066] Such instances will be apparent to the skilled person, and theindication G195GG and corresponding indications for this type ofinsertions are thus meant to comprise such equivalent degenerateindications.

[0067] Filling a Gap:

[0068] Where a deletion in an enzyme exists in comparison to thesubtilisin sequence used for the numbering, an insertion in such aposition is indicated as:

[0069] *36Asp or *36D

[0070] for the insertion of an aspartic acid in position 36

[0071] Multiple Modifications

[0072] Variants comprising multiple modifications are separated bypluses, e.g.:

[0073] Arg170Tyr+Gly195Glu or R170Y+G195E

[0074] representing modifications in positions 170 and 195 substitutingtyrosine and glutamic acid for arginine and glycine, respectively,

[0075] or e.g. Tyr167{Gly,Ala,Ser,Thr}+Arg170{Gly,Ala,Ser,Thr}designates the variants

[0076] Tyr167Gly+Arg170Gly, Tyr167Gly+Arg170Ala,

[0077] Tyr167Gly+Arg170Ser, Tyr167Gly+Arg170Thr,

[0078] Tyr167Ala+Arg170Gly, Tyr167Ala+Arg170Ala,

[0079] Tyr167Ala+Arg170Ser, Tyr167Ala+Arg170Thr,

[0080] Tyr167Ser+Arg170Gly, Tyr167Ser+Arg170Ala,

[0081] Tyr167Ser+Arg170Ser, Tyr167Ser+Arg170Thr,

[0082] Tyr167Thr+Arg170Gly, Tyr167Thr+Arg170Ala,

[0083] Tyr167Thr+Arg170Ser, and Tyr167Thr+Arg170Thr.

[0084] This nomenclature is particular relevant relating tomodifications aimed at substituting, replacing, inserting or deletingamino acid residues having specific common properties, such as residuesof positive charge (K, R, H), negative charge (D, E), or conservativeamino acid modification(s) of e.g.Tyr167{Gly,Ala,Ser,Thr}+Arg170{Gly,Ala,Ser,Thr}, which signifiessubstituting a small amino acid for another small amino acid. Seesection “Detailed description of the invention” for further details.

[0085] Proteases

[0086] Enzymes cleaving the amide linkages in protein substrates areclassified as proteases, or (interchangeably) peptidases (see Walsh,1979, Enzymatic Reaction Mechanisms. W. H. Freeman and Company, SanFrancisco, Chapter 3).

[0087] Numbering of Amino Acid Positions/Residues

[0088] Unless otherwise stated the amino acid numbering used hereincorrespond to that of the subtilase BPN′ (BASBPN) sequence. For furtherdescription of the BPN′ sequence see Siezen et al., Protein Engng. 4(1991) 719-737 and FIG. 1.

[0089] Serine Proteases

[0090] A serine protease is an enzyme which catalyzes the hydrolysis ofpeptide bonds, and in which there is an essential serine residue at theactive site (White, Handler and Smith, 1973 “Principles ofBiochemistry,” Fifth Edition, McGraw-Hill Book Company, NY, pp.271-272).

[0091] The bacterial serine proteases have molecular weights in the20,000 to 45,000 Dalton range. They are inhibited bydiisopropylfluorophosphate. They hydrolyze simple terminal esters andare similar in activity to eukaryotic chymotrypsin, also a serineprotease. A more narrow term, alkaline protease, covering a sub-group,reflects the high pH optimum of some of the serine proteases, from pH9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41711-753).

[0092] Subtilases

[0093] A sub-group of the serine proteases tentatively designatedsubtilases has been proposed by Siezen et al., Protein Engng. 4 (1991)719-737. They are defined by homology analysis of more than 40 aminoacid sequences of serine proteases previously referred to assubtilisin-like proteases. A subtilisin was previously defined as aserine protease produced by Gram-positive bacteria or fungi, andaccording to Siezen et al. now is a subgroup of the subtilases. A widevariety of subtilases have been identified, and the amino acid sequencesof a number of subtilases have been determined. For a more detaileddescription of such subtilases and their amino acid sequences referenceis made to Siezen et al. and FIG. 1 herein.

[0094] One subgroup of the subtilases, I-S1, comprises the “classical”subtilisins, such as subtilisin 168, subtilisin BPN′, subtilisinCarlsberg (ALCALASE®, NOVO NORDISK A/S), and subtilisin DY.

[0095] A further subgroup of the subtilases, I-S2, is recognized bySiezen et al. (supra). Sub-group I-S2 proteases are described as highlyalkaline subtilisins and comprise enzymes such as subtilisin PB92(MAXACAL®, Gist-Brocades NV), subtilisin 309 (SAVINASE®, NOVO NORDISKA/S), subtilisin 147 (ESPERASE®, NOVO NORDISK A/S), and alkalineelastase YaB.

[0096] “SAVINASE®”

[0097] SAVINASE® is marketed by NOVO NORDISK A/S. It is subtilisin 309from B. lentus and differs from BABP92 only in one position (N87S, seeFIG. 1 herein). SAVINASE® has the amino acid sequence designated BLSAVI(see FIG. 1 herein).

[0098] Parent Subtilase

[0099] The term “parent subtilase” is a subtilase defined according toSiezen et al. (Protein Engineering 4:719-737 (1991)). For furtherdetails see description of “SUBTILASES” immediately above. A parentsubtilase may also be a subtilase isolated from a natural source,wherein subsequent modification have been made while retaining thecharacteristic of a subtilase.

[0100] Alternatively the term “parent subtilase” may be termed“wild-type subtilase”.

[0101] Modification(s) of a Subtilase Variant

[0102] The term “modification(s)” used in connection withmodification(s) of a subtilase variant as discussed herein is defined toinclude chemical modification as well as genetic manipulation. Themodification(s) can be by substitution, deletion and/or insertions in orat the amino acid(s) of interest.

[0103] Subtilase Variant

[0104] In the context of this invention, the term subtilase variant ormutated subtilase means a subtilase that has been produced by anorganism which is expressing a mutant gene derived from a parentmicroorganism which possessed an original or parent gene and whichproduced a corresponding parent enzyme, the parent gene having beenmutated in order to produce the mutant gene from which said mutatedsubtilase protease is produced when expressed in a suitable host.

[0105] Homologous Subtilase Sequences

[0106] Specific active site loop regions, and amino acid insertions insaid loops of the subtilase SAVINASE® are identified for modificationherein to obtain a subtilase variant of the invention.

[0107] However, the invention is not limited to modifications of thisparticular subtilase, but extend to other parent (wild-type) subtilases,which have a homologous primary structure to that of SAVINASE®.

[0108] In order to identify other homologous subtilases, within thescope of this invention, an alignment of said subtilase(s) to a group ofpreviously aligned subtilases is performed keeping the previousalignment constant. A comparison to 18 highly conserved residues insubtilases is performed. The 18 highly conserved residues are shown intable I (see Siezen et al. for further details relating to saidconserved residues). TABLE I 18 highly conserved residues in subtilasesPosition: Conserved residue 23 G 32 D 34 G 39 H 64 H 65 G 66 T 70 G 83 G125 S 127 G 146 G 154 G 155 N 219 G 220 T 221 S 225 P

[0109] After aligning allowing for necessary insertions and deletions inorder to maintain the alignment suitable homologous active site loopregions are identified. Said homologous residues can then be modifiedaccording to the invention.

[0110] Using the CLUSTALW (version 1.7, June 1997) computer alignmentprogram (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994)Nucleic Acids Research, 22:4673-4680.), using default alignmentparameters, alignment of a given subtilase to a group of previouslyaligned subtilases is achieved using the Profile alignments option inthe program. For a given subtilase to be within the scope of theinvention, preferably 100% of the 18 highly conserved residues should beconserved. However, alignment of greater than or equal to 17 out of the18 residues, or as little as 16 of said conserved residues is alsoadequate to identify homologous residues. Conservation of the, insubtilases, catalytic triad Asp32/His64/Ser221 should be maintained.

[0111] An alignment of 10 subtilases as defined is shown in FIG. 1.

[0112] Further in said process to identify a homologous parent(wild-type) subtilase within the scope of the invention, the 18conserved residues above relates to the parent (wild-type) primarysequence of said homologous parent subtilase. In other words, if aparent subtilase has been modified in any of said 18 conserved residuesabove, it is the original parent wild-type sequence in said 18 conservedresidues, which determines whether or not both the original parentsubtilase and a possible variant of said parent subtilase, which ismodified in any of said 18 conserved residues above, is a homologoussubtilase within the scope of the present invention.

[0113] Based on this description it is routine for a person skilled inthe art to identify suitable homologous subtilases and correspondinghomologous active site loop regions, which can be modified according tothe invention.

[0114] Wash Performance

[0115] The ability of an enzyme to catalyze the degradation of variousnaturally occurring substrates present on the objects to be cleanedduring e.g. wash is often referred to as its washing ability,wash-ability, detergency, or wash performance. Throughout thisapplication the term wash performance will be used to encompass thisproperty.

[0116] Isolated DNA Sequence

[0117] The term “isolated”, when applied to a DNA sequence molecule,denotes that the DNA sequence has been removed from its natural geneticmilieu and is thus free of other extraneous or unwanted codingsequences, and is in a form suitable for use within geneticallyengineered protein production systems. Such isolated molecules are thosethat are separated from their natural environment and include cDNA andgenomic clones. Isolated DNA molecules of the present invention are freeof other genes with which they are ordinarily associated, but mayinclude naturally occurring 5′and 3′ untranslated regions such aspromoters and terminators. The identification of associated regions willbe evident to one of ordinary skill in the art (see for example, Dynanand Tijan, Nature 316:774-78, 1985). The term “an isolated DNA sequence”may alternatively be termed “a cloned DNA sequence”.

[0118] Isolated Protein

[0119] When applied to a protein, the term “isolated” indicates that theprotein is found in a condition other than its native environment. In apreferred form, the isolated protein is substantially free of otherproteins, particularly other homologous proteins (i.e. “homologousimpurities” (see below)). An isolated protein is more than 10% pure,preferably more than 20% pure, more preferably more than 30% pure, asdetermined by SDS-PAGE. Further it is preferred to provide the proteinin a highly purified form, i.e., more than 40% pure, more than 60% pure,more than 80% pure, more preferably more than 95% pure, and even morepreferably more than 99% pure, as determined by SDS-PAGE.

[0120] The term “isolated protein” may alternatively be termed “purifiedprotein”.

[0121] Homologous Impurities

[0122] The term “homologous impurities” means any impurity (e.g. anotherpolypeptide than the polypeptide of the invention) which originate fromthe homologous cell where the polypeptide of the invention is originallyobtained from.

[0123] Obtained From

[0124] The term “obtained from” as used herein in connection with aspecific microbial source, means that the polynucleotide and/orpolypeptide produced by the specific source, or by a cell in which agene from the source have been inserted.

[0125] Substrate

[0126] The term “Substrate” used in connection with a substrate for aprotease should be interpreted in its broadest form as comprising acompound containing at least one peptide bond susceptible to hydrolysisby a protease.

[0127] Product

[0128] The term “product” used in connection with a product derived froma protease enzymatic reaction should in the context of this invention beinterpreted to include the products of a hydrolysis reaction involving asubtilase protease. A product may be the substrate in a subsequenthydrolysis reaction.

BRIEF DESCRIPTION OF THE DRAWING

[0129]FIGS. 1A and 1B show an alignment of 10 homologous subtilases,which are aligned to the above mentioned 18 highly conserved residues insubtilases, wherein

[0130] BASBPN denotes the Bacillus amyloliquefaciens subtilisin BPN′ setforth in SEQ ID NO: 4,

[0131] BLS147 denotes the Bacillus lentus subtilisin 147 set forth inSEQ ID NO: 5,

[0132] BYSYAB denotes the Bacillus subtilisin YAB set forth in SEQ IDNO: 6,

[0133] BAPBP92 denotes the Bacillus alcalophilus subtilisin PB92 setforth in SEQ ID NO: 7,

[0134] BSSDY denotes the Bacillus subtilis subtilisin DY set forth inSEQ ID NO: 8,

[0135] TVTHER denotes the Thermoactinomyces vulgaris subtilisin setforth in SEQ ID NO: 9,

[0136] BLSAVI denotes the Bacillus lentus subtilisin 309 set forth inSEQ ID NO: 10,

[0137] BSSPI denotes the Bacillus subtilis subtilisin set forth in SEQID NO: 11,

[0138] BSEPR denotes the Bacillus subtilis subtilisin set forth in SEQID NO: 12, and

[0139] JP170 denotes the Bacillus subtilis subtilisin set forth in SEQID NO: 13.

[0140] The 18 highly conserved residues are highlighted in bold. Allshown subtilases, except JP170, have 100% identity in said conservedresidues. JP170 has an asparagine “N” in position 146 instead of theconserved glycine residue “G”.

[0141]FIG. 2 shows an alignment of three Savinase variants of theinvention with the alignment shown in FIG. 1. Each of the variants37.03, 37.04 and 37.06 (SEQ ID NOS: 14-16) is aligned individually withthe alignment of FIG. 1. All three variants are shown in one figure forbrevity.

[0142]FIG. 3 shows the three-dimensional structure of Savinase (Proteindata bank (PDB) entry 1SVN). In this figure the active site loops ofinterest herein are indicated.

DETAILED DESCRIPTION OF THE INVENTION

[0143] Subtilase Enzymes With Improved Wash Performance:

[0144] The subtilases of the invention are generally described in thesection Summary of the Invention.

[0145] A subtilase of the first aspect of the invention may be a parentwild-type subtilase identified in nature.

[0146] Such a parent wild-type subtilase may be specifically screenedfor by standard techniques known in the art.

[0147] One preferred way of doing this may be by specifically PCRamplify DNA regions known to encode active site loops in subtilases fromnumerous different microorganism, preferably different Bacillus strains.

[0148] Subtilases are a group of conserved enzymes, in the sense thattheir DNA and amino acid sequences are homologous. Accordingly it ispossible to construct relatively specific primers flanking active siteloops.

[0149] E.g. by investigating alignment of different subtilases (see e.g.Siezen et al. Protein Science 6:501-523 (1997)), it is routine work fora person skilled in the art to construct PCR primers flanking e.g. theactive site loop corresponding to active site loop between amino acidresidue 95 to 103 in BLSAVI. Using those PCR primers to amplify DNA froma number of different microorganisms, preferably different Bacillusstrains, followed by DNA sequencing said amplified PCR fragments, itwill be possible to identify those strains which produce subtilases,which comprises a longer, as compared to BLSAVI, active site regioncorresponding the active site region of 95-103 in BLSAVI. Havingidentified the strain and a partial DNA sequence of such a subtilase ofinterest, it is routine work for a person skilled in the art to completecloning, expression and purification of such a subtilase of interest.

[0150] However, it is envisaged that a subtilase enzyme of the inventionpredominantly is a variant of a parent subtilase.

[0151] Accordingly, an embodiment of the invention relates to a isolatedsubtilase enzyme according to the first aspect of the invention, whereinsaid subtilase enzyme is a constructed variant, wherein said variantcomprises at least one insertion of at least one amino acid within atleast one of the active site loops according to the first aspect of theinvention.

[0152] A subtilase enzyme of the invention exhibits improved washperformance, as compared to BLSAVI (Savinase®), in a detergent.Different commercial subtilase protease products will exhibit adifferent wash performance in different kinds of detergent compositions.A subtilase of the invention exhibits improved wash performance, ascompared to BLSAVI, in a majority of different kinds of detergentcompositions.

[0153] Preferably a subtilase enzyme of the invention exhibits improvedwash performance, as compared to BLSAVI, in the detergent compositionshown in working example 3 herein (vide infra).

[0154] In order to identify whether or not a given subtilase amino acidsequence (independent of whether said subtilase sequence is a wild typesubtilase sequence isolated from nature or a subtilase variant sequence)is within the scope of a subtilase sequence of the invention, thefollowing steps may to be performed:

[0155] i) identify if said subtilase sequence is at least 40%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 95% identical to the aminoacid sequence from position 1 to position 275 of subtilase BLSAVI (inBASBPN numbering);

[0156] ii) if step i) is fulfilled, perform an alignment of saidsubtilase sequence to the previously defined alignment of subtilasesspecified in FIG. 1 (see section “Definitions herein (vide supra) inorder to see how this alignment preferably must be performed);

[0157] iii) based on the alignment performed in step ii) identify theactive site loops, in said subtilase sequence, which correspond to theactive site loop regions in BLSAVI, wherein said active site loops arespecified as (in BASBPN as (in BASBPN numbering)

[0158] (a) the region (both of the end amino acids included) betweenamino acid residue from 33 to 43;

[0159] (b) the region (both of the end amino acids included) betweenamino acid residue 95 to 103;

[0160] (c) the region (both of the end amino acids included) betweenamino acid residue 125 to 132;

[0161] (d) the region (both of the end amino acids included) betweenamino acid residue 153 to 173;

[0162] (e) the region (both of the end amino acids included) betweenamino acid residue 181 to 195;

[0163] (f) the region (both of the end amino acids included) betweenamino acid residue 202 to 204; and

[0164] (g) the region (both of the end amino acids included) betweenamino acid residue 218 to 219;

[0165] iv) identify whether or not one or more of the active site loopsin said subtilase sequence, identified in step iii) is longer than thecorresponding active site loop in BLSAVI.

[0166] If one the criteria in step iv) above is fulfilled the givensubtilase sequence is a subtilase sequence within the scope of thepresent invention.

[0167] The identity specified in step i) above between a subtilase ofthe invention and BLSAVI is calculated as described immediately below.

[0168] Identity of Amino Acid Sequences of a Subtilase of the Inventionto BLSAVI.

[0169] The polypeptide identity referred to above is determined as thedegree of identity between the two sequences indicating a derivation ofthe first sequence from the second. The identity may suitably bedetermined by means of computer programs known in the art such as GAPprovided in the GCG program package (Program Manual for the WisconsinPackage, Version 8, August 1994, Genetics Computer Group, 575 ScienceDrive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D.,(1970), Journal of Molecular Biology, 48, 443-453. Using GAP with thefollowing settings for polypeptide sequence comparison: GAP creationpenalty of 3.0 and GAP extension penalty of 0.1, the mature part of asubtilase amino acid sequence of the invention exhibits a degree ofidentity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or even 95% with the mature part of the amino acid sequence ofBLSAVI from position 1 to position 275 (in BASBPN numbering).Accordingly, the identity will be defined as the number of identicalresidues divided by 269 (BLSSAVI mature part has 269 amino acids.)

[0170] The alignment to be performed in step ii) above is performed asdescribed immediately below:

[0171] Alignment of a Subtilase Amino Acid of the Invention to aPreviously Defined Alignment of Homologous Subtilase Sequences (Step Ii)Above), and Identification of Suitable Homologous Active Site Loops, inSaid Subtilase, Which Correspond to the Active Site Loop Regions inBLSAVI (Step iii) Above).

[0172] In order to identify other homologous subtilases, within thescope of this invention, an alignment of said subtilase(s) to a group ofpreviously aligned subtilases is performed keeping the previousalignment constant (step ii) above).

[0173] Using the CLUSTALW (version 1.7, June 1997) computer alignmentprogram (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994)Nucleic Acids Research, 22:4673-4680), using default alignmentparameters, alignment of a given subtilase to a group of previouslyaligned subtilases is achieved using the Profile alignments option inthe program. Conservation of the, in subtilases, catalytic triadAsp32/His64/Ser221 should be maintained.

[0174] The above defined alignment of a group of subtilases is shownFIG. 1.

[0175] After aligning allowing for necessary insertions and deletions inorder to maintain the alignment suitable homologous active site loops,in said subtilase of the invention are identified as described in stepiii) above.

[0176] Based on this description it is routine for a person skilled inthe art to identify suitable homologous subtilases and correspondinghomologous suitable homologous active site loops, in said subtilase.

[0177] A preferred active site loop of a subtilase of the invention asdescribed are the loops defined as

[0178] (b) the region (both of the end amino acids included) betweenamino acid residue 95 to 103 is at least 10 amino acids long (i.e. atleast one amino acid insertion, as compared to BLSAVI); and

[0179] (c) the region (both of the end amino acids included) betweenamino acid residue 125 to 132 is at least 9 amino acids long (i.e. atleast one amino acid insertion, as compared to BLSAVI).

[0180] A subtilase variant may be constructed by standard techniquesknown in the art such as by site-directed/random mutagenesis or by DNAshuffling of different subtilase sequences. See section “PRODUCING ASUBTILASE VARIANT” and Material and methods herein (vide infra) forfurther details.

[0181] In further embodiments the invention relates to

[0182] 1. an isolated subtilase enzyme according to the invention,wherein at least one of said inserted amino acid residue is chosen fromthe group comprising: T, G, A, and S;

[0183] 2. an isolated subtilase enzyme according to the invention,wherein at least one of said inserted amino acid residue is chosen fromthe group of charged amino acid residues comprising: D, E, H, K, and R,more preferably D, E, K and R;

[0184] 3. an isolated subtilase enzyme according to the invention,wherein at least one of said inserted amino acid residue is chosen fromthe group of hydrophilic amino acid residues comprising: C, N, Q, S andT, more preferably N, Q, S and T;

[0185] 4. an isolated subtilase enzyme according to the invention,wherein at least one of said inserted amino acid residue is chosen fromthe group of small hydrophobic amino acid residues comprising: A, G andV; or

[0186] 5. an isolated subtilase enzyme according to the invention,wherein at least one of said inserted amino acid residue is chosen fromthe group of large hydrophilic amino acid residues comprising: F, I, L,M, P, W and Y, more preferably F, I, L, M, and Y.

[0187] In a further embodiment, the invention relates to an isolatedsubtilase enzyme according to the invention, wherein said insertion, inat least one of the active site loops, comprises at least two aminoacids, as compared to the corresponding active site loop in BLSAVI.

[0188] In a further embodiment, the invention relates to an isolatedsubtilase enzyme according to the invention, wherein the subtilaseenzyme is comprising at least one insertion, chosen from the groupcomprising (in BASBPN numbering):

[0189] G97GASG;

[0190] G97GM; and

[0191] G97GAS; and

[0192] an isolated subtilase enzyme according to the invention, whereinthe subtilase enzyme comprises at least one insertion/modification,chosen from the group comprising (in BASBPN numbering):

[0193] 37.03: G97GASG+A98S+S99G+G100A+S101A;

[0194] 37.06: G97GAA+A98S+S99G+S101T; and

[0195] 37.04: G97GAS+A98S+S99G.

[0196] An alignment of residues 91 to 107 of the three latter variantsis shown in FIG. 2.

[0197] It is well known in the art that substitution of one amino acidto a similar conservative amino acid most often only provide minorchanges in the characteristic of the enzyme.

[0198] Table II below list groups of conservative amino acids. TABLE IIConservative amino acid substitutions Basic: R = arginine K = lysine H =histidine Acidic: E = glutamic acid D = aspartic acid Polar: Q =glutamine N = asparagine Hydrophobic: L = leucine I = isoleucine V =valine M = methionine Aromatic: F = phenylalanine W = tryptophan Y =tyrosine Small: G = glycine A = alanine S = serine T = threonine

[0199] Accordingly, subtilase variants such as G97GGG+A98S+S99G, areexpected to exhibit a similar wash-performance improvement as thevariant G97GAA+A98S+S99G. See e.g. working examples herein for aspecific wash performance test of said G97GM+A98S+S99G variant.

[0200] Based on the disclosed and in particular the exemplifiedsubtilase variants herein, it is routine work, for a person skilled inthe art, to identify further suitable conservative modification(s), ofin particular said exemplified variants, in order to obtain a subtilasevariant with improved wash-performance, according to all aspects andembodiments of the invention.

[0201] In embodiments of the invention, the subtilases of interest arepreferably those belonging to the subgroups I-S1 and I-S2 elating tosubgroup I-S1 a preferred parent subtilase is chosen from the groupcomprising ABSS168, BASBPN, BSSDY, and BLSCAR or functional variantsthereof having retained the characteristic of sub-group I-S1.

[0202] Relating to subgroup I-S2 a preferred parent subtilase is chosenfrom the group comprising BLS147, BLSAVI, BLS309, BAPB92, TVTHER ANDBYSYAB or functional variants thereof having retained the characteristicof sub-group I-S2.

[0203] In particular said parent subtilase is BLSAVI (SAVINASE® NOVONORDISK A/S) or subtilases having an identity of 95% or more thereto,and a preferred subtilase variant of the invention is accordingly avariant of SAVINASE® or subtilases having an identity of 95% or morethereto.

[0204] The present invention also comprises any one or moremodifications in the above mentioned positions in combination with anyother modification to the amino acid sequence of the parent enzyme.Especially combinations with other modifications known in the art toprovide improved properties to the enzyme are envisaged. The artdescribes a number of subtilase variants with different improvedproperties and a number of those are mentioned in the “Background of theinvention” section herein (vide supra). Those references are disclosedhere as references to identify a subtilase variant, which advantageouslycan be combined with a subtilase variant of the invention.

[0205] Such combinations comprise the positions: 222 (improve oxidationstability), 218 (improves thermal stability), substitutions in theCa-binding sites stabilizing the enzyme, e.g. position 76, and manyother apparent from the prior art.

[0206] In further embodiments a subtilase variant of the invention mayadvantageously be combined with one or more modification(s) in any ofthe positions:

[0207] 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 206, 218,222, 224, 235 and 274.

[0208] Specifically the following BLS309 and BAPB92 variants areconsidered appropriate for combination:

[0209] K27R, *36D, S57P, N76D, S87N, G97N, S101G, S103A, V104A, V1041,V104N, V104Y, H120D,

[0210] N123S, Y167, R170, Q206E, N218S, M222A, M222S, T224S, K235L andT274A.

[0211] Furthermore variants comprising any of the variantsK27R+V104Y+N123S+T274A, N76D+S103A+V1041, N76D+V104A, S87N+S101G+V104N,S101 G+V104N, or other combinations of these mutations (K27R, N76D,S101G, V104A, V104N, V104Y, N123S, T274A), in combination with any oneor more of the modification(s) mentioned above exhibit improvedproperties.

[0212] Even further subtilase variants of the main aspect(s) of theinvention are preferably combined with one or more modification(s) inany of the positions 129, 131, 133 and 194, preferably as 129K, 131H,133P, 133D and 194P modifications, and most preferably as P129K, P131H,A133P, A133D and A194P modifications. Any of those modification(s) givea higher expression level of a subtilase variant of the invention.

[0213] Accordingly, an even further embodiment of the invention relatesto a variant according to the invention, wherein said modification ischosen from the group comprising:

[0214] Y167A+R170S+A194P

[0215] Y167A+R170L+A194P

[0216] Y167A+R170N+A194P

[0217] Y167A+R170S+P129K

[0218] Y167A+R170L+P129K

[0219] Y167A+R170N+P129K

[0220] Y167A+R170S+P131H

[0221] Y167A+R170L+P131H

[0222] Y167A+R170N+P131H

[0223] Y167A+R170S+A133P

[0224] Y167A+R170L+A133P

[0225] Y167A+R170N+A133P

[0226] Y167A+R170S+A133D

[0227] Y167A+R170L+A133D

[0228] Y167A+R170N+A133D

[0229] Producing A Subtilase Variant

[0230] Many methods for cloning a subtilase of the invention and forintroducing insertions into genes (e.g. subtilase genes) are well knownin the art.

[0231] In general standard procedures for cloning of genes andintroducing insertions (random and/or site directed) into said genes maybe used in order to obtain a subtilase variant of the invention. Forfurther description of suitable techniques reference is made to workingexamples herein (vide infra) and (Sambrook et al. (1989) Molecularcloning: A laboratory manual, Cold Spring Harbor lab., Cold SpringHarbor, N.Y.; Ausubel, F. M. et al. (eds.) “Current protocols inMolecular Biology”. John Wiley and Sons, 1995; Harwood, C. R., andCutting, S. M. (eds.) “Molecular Biological Methods for Bacillus”. JohnWiley and Sons, 1990); and WO 96/34946.

[0232] Further a subtilase variant of the invention may be constructedby standard techniques of DNA shuffling of different subtilase genes (WO95/22625; Stemmer WPC, Nature 370:389-91 (1994)). DNA shuffling of e.g.Savinase® with one or more partial subtilase sequences identified innature to comprise longer than Savinase® active site loops regions, willafter subsequent screening for improved wash performance variants,provide subtilase variants according to the invention.

[0233] Expression Vectors

[0234] A recombinant expression vector comprising a DNA constructencoding the enzyme of the invention may be any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e. a vector which exists is as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome in part or inits entirety and replicated together with the chromosome(s) into whichit has been integrated.

[0235] The vector is preferably an expression vector in which the DNAsequence encoding the enzyme of the invention is operably linked toadditional segments required for transcription of the DNA. In general,the expression vector is derived from plasmid or viral DNA, or maycontain elements of both. The term, “operably linked” indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in a promoter andproceeds through the DNA sequence coding for the enzyme.

[0236] The promoter may be any DNA sequence which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.

[0237] Examples of suitable promoters for use in bacterial host cellsinclude the promoter of the Bacillus stearothermophilus maltogenicamylase gene, the Bacillus licheniformis alpha-amylase gene, theBacillus amyloliquefaciens alpha-amylase gene, the Bacillus subtilisalkaline protease gene, or the Bacillus pumilus xylosidase gene, or thephage Lambda PR or PL promoters or the E. coli lac, trp or tacpromoters.

[0238] The DNA sequence encoding the enzyme of the invention may also,if necessary, be operably connected to a suitable terminator.

[0239] The recombinant vector of the invention may further comprise aDNA sequence enabling the vector to replicate in the host cell inquestion.

[0240] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, or a geneencoding resistance to e.g. antibiotics like kanamycin, chloramphenicol,erythromycin, tetracycline, spectinomycine, or the like, or resistanceto heavy metals or herbicides.

[0241] To direct an enzyme of the present invention into the secretorypathway of the host cells, a secretory signal sequence (also known as aleader sequence, pre-pro sequence or pre sequence) may be provided inthe recombinant vector. The secretory signal sequence is joined to theDNA sequence encoding the enzyme in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe enzyme. The secretory signal sequence may be that normallyassociated with the enzyme or may be from a gene encoding anothersecreted protein.

[0242] The procedures used to ligate the DNA sequences coding for thepresent enzyme, the promoter and optionally the terminator and/orsecretory signal sequence, respectively, or to assemble these sequencesby suitable PCR amplification schemes, and to insert them into suitablevectors containing the information necessary for replication orintegration, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., op.cit.).

[0243] Host Cell

[0244] The DNA sequence encoding the present enzyme introduced into thehost cell may be either homologous or heterologous to the host inquestion. If homologous to the host cell, i.e. produced by the host cellin nature, it will typically be operably connected to another promotersequence or, if applicable, another secretory signal sequence and/orterminator sequence than in its natural environment. The term“homologous” is intended to include a DNA sequence encoding an enzymenative to the host organism in question. The term “heterologous” isintended to include a DNA sequence not expressed by the host cell innature. Thus, the DNA sequence may be from another organism, or it maybe a synthetic sequence.

[0245] The host cell into which the DNA construct or the recombinantvector of the invention is introduced may be any cell which is capableof producing the present enzyme and includes bacteria, yeast, fungi andhigher eukaryotic cells.

[0246] Examples of bacterial host cells which, on cultivation, arecapable of producing the enzyme of the invention are gram-positivebacteria such as strains of Bacillus, such as strains of B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus, B. megatherium or B. thuringiensis, or strains of Streptomyces,such as S. lividans or S. murinus, or gram-negative bacteria such asEcherichia coli. The transformation of the bacteria may be effected byprotoplast transformation, electroporation, conjugation, or by usingcompetent cells in a manner known per se (cf. Sambrook et al., supra).

[0247] When expressing the enzyme in bacteria such as E. coli, theenzyme may be retained in the cytoplasm, typically as insoluble granules(known as inclusion bodies), or may be directed to the periplasmic spaceby a bacterial secretion sequence. In the former case, the cells arelysed and the granules are recovered and denatured after which theenzyme is refolded by diluting the denaturing agent. In the latter case,the enzyme may be recovered from the periplasmic space by disrupting thecells, e.g. by sonication or osmotic shock, to release the contents ofthe periplasmic space and recovering the enzyme.

[0248] When expressing the enzyme in gram-positive bacteria such asBacillus or Streptomyces strains, the enzyme may be retained in thecytoplasm, or may be directed to the extracellular medium by a bacterialsecretion sequence. In the latter case, the enzyme may be recovered fromthe medium as described below.

[0249] Method of Producing Subtilase

[0250] The present invention provides a method of producing an isolatedenzyme according to the invention, wherein a suitable host cell, whichhas been transformed with a DNA sequence encoding the enzyme, iscultured under conditions permitting the production of the enzyme, andthe resulting enzyme is recovered from the culture.

[0251] When an expression vector comprising a DNA sequence encoding theenzyme is transformed into a heterologous host cell it is possible toenable heterologous recombinant production of the enzyme of theinvention.

[0252] Thereby it is possible to make a highly purified subtilasecomposition, characterized in being free from homologous impurities.

[0253] In this context, homologous impurities mean any impurities (e.g.other polypeptides than the enzyme of the invention) which originatefrom the homologous cell where the enzyme of the invention is originallyobtained from.

[0254] The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed subtilase may conveniently be secreted into the culture mediumand may be recovered therefrom by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulfate, followed by chromatographic procedures such asion exchange chromatography, affinity chromatography, or the like.

[0255] Use of a Subtilase Variant of the Invention

[0256] A subtilase protease variant of the invention may be used for anumber of industrial applications, in particular within the detergentindustry.

[0257] Further the invention relates to an enzyme composition, whichcomprises a subtilase variant of the invention.

[0258] A summary of preferred industrial applications and correspondingpreferred enzyme, compositions are described below.

[0259] This summary is not in any way intended to be a complete list ofsuitable applications of a subtilase variant of the invention. Asubtilase variant of the invention may be used in other industrialapplications known in the art to include use of a protease, inparticular a subtilase.

[0260] Detergent Compositions Comprising the Mutant Enzymes

[0261] The present invention comprises the use of the mutant enzymes ofthe invention in cleaning and detergent compositions and suchcompositions comprising the mutant subtilisin enzymes. Such cleaning anddetergent compositions are well described in the art and reference ismade to WO 96/34946; WO 97/07202; WO 95/30011 for further description ofsuitable cleaning and detergent compositions.

[0262] Further reference is made to workings example(s) herein showingwash performance improvements for a number of subtilase variants of theinvention.

[0263] Surfactant System

[0264] The detergent compositions according to the present inventioncomprise a surfactant system, wherein the surfactant can be selectedfrom nonionic and/or anionic and/or cationic and/or ampholytic and/orzwitterionic and/or semi-polar surfactants.

[0265] The surfactant is typically present at a level from 0.1% to 60%by weight.

[0266] The surfactant is preferably formulated to be compatible withenzyme components present in the composition. In liquid or gelcompositions the surfactant is most preferably formulated in such a waythat it promotes, or at least does not degrade, the stability of anyenzyme in these compositions.

[0267] Preferred systems to be used according to the present inventioncomprise as a surfactant one or more of the nonionic and/or anionicsurfactants described herein.

[0268] Polyethylene, polypropylene, and polybutylene oxide condensatesof alkyl phenols are suitable for use as the nonionic surfactant of thesurfactant systems of the present invention, with the polyethylene oxidecondensates being preferred. These compounds include the condensationproducts of alkyl phenols having an alkyl group containing from about 6to about 14 carbon atoms, preferably from about 8 to about 14 carbonatoms, in either a straight chain or branched-chain configuration withthe alkylene oxide. In a preferred embodiment, the ethylene oxide ispresent in an amount equal to from about 2 to about 25 moles, morepreferably from about 3 to about 15 moles, of ethylene oxide per mole ofalkyl phenol. Commercially available nonionic surfactants of this typeinclude Igepal™ CO-630, marketed by the GAF Corporation; and Triton™X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company.These surfactants are commonly referred to as alkylphenol alkoxylates(e.g., alkyl phenol ethoxylates).

[0269] The condensation products of primary and secondary aliphaticalcohols with about 1 to about 25 moles of ethylene oxide are suitablefor use as the nonionic surfactant of the nonionic surfactant systems ofthe present invention. The alkyl chain of the aliphatic alcohol caneither be straight or branched, primary or secondary, and generallycontains from about 8 to about 22 carbon atoms. Preferred are thecondensation products of alcohols having an alkyl group containing fromabout 8 to about 20 carbon atoms, more preferably from about 10 to about18 carbon atoms, with from about 2 to about 10 moles of ethylene oxideper mole of alcohol. About 2 to about 7 moles of ethylene oxide and mostpreferably from 2 to 5 moles of ethylene oxide per mole of alcohol arepresent in said condensation products. Examples of commerciallyavailable nonionic surfactants of this type include Tergitol™ 15-S-9(The condensation product of C₁₁-C₁₅ linear alcohol with 9 molesethylene oxide), Tergitol™ 24-L-6 NMW (the condensation product ofC₁₂-C₁₄ primary alcohol with 6 moles ethylene oxide with a narrowmolecular weight distribution), both marketed by Union CarbideCorporation; Neodol™ 45-9 (the condensation product of C₁₄-C₁₅ linearalcohol with 9 moles of ethylene oxide), Neodol™ 23-3 (the condensationproduct of C₁₂-C₁₃ linear alcohol with 3.0 moles of ethylene oxide),Neodol™ 45-7 (the condensation product of C₁₄-C₁₅ linear alcohol with 7moles of ethylene oxide), Neodol™ 45-5 (the condensation product ofC₁₄-C₁₅ linear alcohol with 5 moles of ethylene oxide) marketed by ShellChemical Company, Kyro™ EOB (the condensation product of C₁₃-C₁₅ alcoholwith 9 moles ethylene oxide), marketed by The Procter & Gamble Company,and Genapol LA 050 (the condensation product of C₁₂-C₁₄ alcohol with 5moles of ethylene oxide) marketed by Hoechst. Preferred range of HLB inthese products is from 8-11 and most preferred from 8-10.

[0270] Also useful as the nonionic surfactant of the surfactant systemsof the present invention are alkylpolysaccharides disclosed in U.S. Pat.No. 4,565,647, having a hydrophobic group containing from about 6 toabout 30 carbon atoms, preferably from about 10 to about 16 carbon atomsand a polysaccharide, e.g. a polyglycoside, hydrophilic group containingfrom about 1.3 to about 10, preferably from about 1.3 to about 3, mostpreferably from about 1.3 to about 2.7 saccharide units. Any reducingsaccharide containing 5 or 6 carbon atoms can be used, e.g., glucose,galactose and galactosyl moieties can be substituted for the glucosylmoieties (optionally the hydrophobic group is attached at the 2-, 3-,4-, etc. positions thus giving a glucose or galactose as opposed to aglucoside or galactoside). The intersaccharide bonds can be, e.g.,between the one position of the additional saccharide units and the 2-,3-, 4-, and/or 6-positions on the preceding saccharide units.

[0271] The preferred alkylpolyglycosides have the formula

R²O(C_(n)H_(2n)O)_(t)(glycosyl)_(x)

[0272] wherein R² is selected from the group consisting of alkyl,alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof inwhich. the alkyl groups contain from about 10 to about 18, preferablyfrom about 12 to about 14, carbon atoms; n is 2 or 3, preferably 2; t isfrom 0 to about 10, preferably 0; and x is from about 1.3 to about 10,preferably from about 1.3 to about 3, most preferably from about 1.3 toabout 2.7. The glycosyl is preferably derived from glucose. To preparethese compounds, the alcohol or alkylpolyethoxy alcohol is formed firstand then reacted with glucose, or a source of glucose, to form theglucoside (attachment at the 1-position). The additional glycosyl unitscan then be attached between their 1-position and the preceding glycosylunits 2-, 3-, 4-, and/or 6-position, preferably predominantly the2-position.

[0273] The condensation products of ethylene oxide with a hydrophobicbase formed by the condensation of propylene oxide with propylene glycolare also suitable for use as the additional nonionic surfactant systemsof the present invention. The hydrophobic portion of these compoundswill preferably have a molecular weight from about 1500 to about 1800and will exhibit water insolubility. The addition of polyoxyethylenemoieties to this hydrophobic portion tends to increase the watersolubility of the molecule as a whole, and the liquid character of theproduct is retained up to the point where the polyoxyethylene content isabout 50% of the total weight of the condensation product, whichcorresponds to condensation with up to about 40 moles of ethylene oxide.Examples of compounds of this type include certain of the commerciallyavailable Pluronic™ surfactants, marketed by BASF.

[0274] Also suitable for use as the nonionic surfactant of the nonionicsurfactant system of the present invention, are the condensationproducts of ethylene oxide with the product resulting from the reactionof propylene oxide and ethylenediamine. The hydrophobic moiety of theseproducts consists of the reaction product of ethylenediamine and excesspropylene oxide, and generally has a molecular weight of from about 2500to about 3000. This hydrophobic moiety is condensed with ethylene oxideto the extent that the condensation product contains from about 40% toabout 80% by weight of polyoxyethylene and has a molecular weight offrom about 5,000 to about 11,000. Examples of this type of nonionicsurfactant include certain of the commercially available Tetronic™compounds, marketed by BASF.

[0275] Preferred for use as the nonionic surfactant of the surfactantsystems of the present invention are polyethylene oxide condensates ofalkyl phenols, condensation products of primary and secondary aliphaticalcohols with from about 1 to about 25 moles of ethylene oxide,alkylpolysaccharides, and mixtures hereof. Most preferred are C₈-C₁₄alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C₈-C₁₈alcohol ethoxylates (preferably C₁₀ avg.) having from 2 to 10 ethoxygroups, and mixtures thereof.

[0276] Highly preferred nonionic surfactants are polyhydroxy fatty acidamide surfactants of the formula

[0277] wherein R¹ is H, or R¹ is C₁₋₄ hydrocarbyl, 2-hydroxyethyl,2-hydroxypropyl or a mixture thereof, R² is C₅₋₃₁ hydrocarbyl, and Z isa polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least3 hydroxyls directly connected to the chain, or an alkoxylatedderivative thereof. Preferably, R¹ is methyl, R² is straight C₁₁₋₁₅alkyl or C₁₆₋₁₈ alkyl or alkenyl chain such as coconut alkyl or mixturesthereof, and Z is derived from a reducing sugar such as glucose,fructose, maltose or lactose, in a reductive amination reaction.

[0278] Highly preferred anionic surfactants include alkyl alkoxylatedsulfate surfactants. Examples hereof are water soluble salts or acids ofthe formula RO(A)_(m)SO3M wherein R is an unsubstituted C₁₀-C-₂₄ alkylor hydroxyalkyl group having a C₁₀-C₂₄ alkyl component, preferably aC₁₂-C₂₀ alkyl or hydroxyalkyl, more preferably C₁₂-C₁₈ alkyl orhydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero,typically between about 0.5 and about 6, more preferably between about0.5 and about 3, and M is H or a cation which can be, for example, ametal cation (e.g., sodium, potassium, lithium, calcium, magnesium,etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylatedsulfates as well as alkyl propoxylated sulfates are contemplated herein.Specific examples of substituted ammonium cations include methyl-,dimethyl, trimethyl-ammonium cations and quaternary ammonium cationssuch as tetramethyl-ammonium and dimethyl piperidinium cations and thosederived from alkylamines such as ethylamine, diethylamine,triethylamine, mixtures thereof, and the like. Exemplary surfactants areC₁₂-C₁₈ alkyl polyethoxylate (1.0) sulfate (C₁₂-C₁₈E(1.0)M), C₁₂-C₁₈alkyl polyethoxylate (2.25) sulfate (C₁₂-C₁₈(2.25)M, and C₁₂-C₁₈ alkylpolyethoxylate (3.0) sulfate (C₁₂-C₁₈E(3.0)M), and C₁₂-C₁₈ alkylpolyethoxylate (4.0) sulfate (C₁₂-C₁₈E(4.0)M), wherein M is convenientlyselected from sodium and potassium.

[0279] Suitable anionic surfactants to be used are alkyl ester sulfonatesurfactants including linear esters of C₈-C₂₀ carboxylic acids (i.e.,fatty acids) which are sulfonated with gaseous SO₃ according to “TheJournal of the American Oil Chemists Society”, 52 (1975), pp. 323-329.Suitable starting materials would include natural fatty substances asderived from tallow, palm oil, etc.

[0280] The preferred alkyl ester sulfonate surfactant, especially forlaundry applications, comprise alkyl ester sulfonate surfactants of thestructural formula:

[0281] wherein R³ is a C₈-C₂₀ hydrocarbyl, preferably an alkyl, orcombination thereof, R⁴ is a C₁-C₆ hydrocarbyl, preferably an alkyl, orcombination thereof, and M is a cation which forms a water soluble saltwith the alkyl ester sulfonate. Suitable salt-forming cations includemetals such as sodium, potassium, and lithium, and substituted orunsubstituted ammonium cations, such as monoethanolamine,diethonolamine, and triethanolamine. Preferably, R³ is C₁₀-C₁₆ alkyl,and R⁴ is methyl, ethyl or isopropyl. Especially preferred are themethyl ester sulfonates wherein R³ is C₁₀-C₁₆ alkyl.

[0282] Other suitable anionic surfactants include the alkyl sulfatesurfactants which are water soluble salts or acids of the formula ROSO₃Mwherein R preferably is a C₁₀-C₂₄ hydrocarbyl, preferably an alkyl orhydroxyalkyl having a C₁₀-C₂₀ alkyl component, more preferably a C₁₂-C₁₈alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metalcation (e.g. sodium, potassium, lithium), or ammonium or substitutedammonium (e.g. methyl-, dimethyl-, and trimethyl ammonium cations andquaternary ammonium cations such as tetramethyl-ammonium and dimethylpiperidinium cations and quaternary ammonium cations derived fromalkylamines such as ethylamine, diethylamine, triethylamine, andmixtures thereof, and the like). Typically, alkyl chains of C₁₂-C₁₆ arepreferred for lower wash temperatures (e.g. below about 50° C.) andC₁₆-C₁₈ alkyl chains are preferred for higher wash temperatures (e.g.above about 50° C.).

[0283] Other anionic surfactants useful for detersive purposes can alsobe included in the laundry detergent compositions of the presentinvention. Theses can include salts (including, for example, sodium,potassium, ammonium, and substituted ammonium salts such as mono-, di-and triethanolamine salts) of soap, C₈-C₂₂ primary or secondaryalkanesulfonates, C₈-C₂₄ olefinsulfonates, sulfonated polycarboxylicacids prepared by sulfonation of the pyrolyzed product of alkaline earthmetal citrates, e.g., as described in British patent specification No.1,082,179, C₈-C₂₄ alkylpolyglycolethersulfates (containing up to 10moles of ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerolsulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxideether sulfates, paraffin sulfonates, alkyl phosphates, isethionates suchas the acyl isethionates, N-acyl taurates, alkyl succinamates andsulfosuccinates, monoesters of sulfosuccinates (especially saturated andunsaturated C₁₂-C₁₈ monoesters) and diesters of sulfosuccinates(especially saturated and unsaturated C₆-C₁₂ diesters), acylsarcosinates, sulfates of alkylpolysaccharides such as the sulfates ofalkylpolyglucoside (the nonionic non-sulfated compounds being describedbelow), branched primary alkyl sulfates, and alkyl polyethoxycarboxylates such as those of the is formula RO(CH₂CH₂O)_(k)—CH₂C00-M+wherein R is a C₈-C₂₂ alkyl, k is an integer from 1 to 10, and M is asoluble salt forming cation. Resin acids and hydrogenated resin acidsare also suitable, such as rosin, hydrogenated rosin, and resin acidsand hydrogenated resin acids present in or derived from tall oil.

[0284] Alkylbenzene sulfonates are highly preferred. Especiallypreferred are linear (straight-chain) alkyl benzene sulfonates (LAS)wherein the alkyl group preferably contains from 10 to 18 carbon atoms.

[0285] Further examples are described in “Surface Active Agents andDetergents” (Vol. I and II by Schwartz, Perrry and Berch). A variety ofsuch surfactants are also generally disclosed in U.S. Pat. No. 3,929,678(column 23, line 58 through column 29, line 23, herein incorporated byreference).

[0286] When included therein, the laundry detergent compositions of thepresent invention typically comprise from about 1% to about 40%,preferably from about 3% to about 20% by weight of such anionicsurfactants.

[0287] The laundry detergent compositions of the present invention mayalso contain cationic, ampholytic, zwitterionic, and semi-polarsurfactants, as well as the nonionic and/or anionic surfactants otherthan those already described herein.

[0288] Cationic detersive surfactants suitable for use in the laundrydetergent compositions of the present invention are those having onelong-chain hydrocarbyl group. Examples of such cationic surfactantsinclude the ammonium surfactants such as alkyltrimethylammoniumhalogenides, and those surfactants having the formula:

[R²(OR³)_(y)][R⁴(OR³)_(y)]₂R⁵N+X—

[0289] wherein R² is an alkyl or alkyl benzyl group having from about 8to about 18 carbon atoms in the alkyl chain, each R³ is selected formthe group consisting of —CH₂CH₂—, —CH₂CH(CH₃)—, —CH₂CH(CH₂OH)—,—CH₂CH₂CH₂—, and mixtures thereof; each R⁴ is selected from the groupconsisting of C₁-C₄ alkyl, C₁-C₄ hydroxyalkyl, benzyl ring structuresformed by joining the two R⁴ groups, —CH₂CHOHCHOHCOR⁶CHOHCH₂OH, whereinR⁶ is any hexose or hexose polymer having a molecular weight less thanabout 1000, and hydrogen when y is not 0; R⁵ is the same as R⁴ or is analkyl chain, wherein the total number of carbon atoms or R² plus R⁵ isnot more than about 18; each y is from 0 to about 10, and the sum of they values is from 0 to about 15; and X is any compatible anion.

[0290] Highly preferred cationic surfactants are the water solublequaternary ammonium compounds useful in the present composition havingthe formula:

R₁R₂R₃R₄N⁺X⁻  (i)

[0291] wherein R₁ is C₈-C₁₆ alkyl, each of R₂, R₃ and R₄ isindependently C₁-C₄ alkyl, C₁-C₄ hydroxy alkyl, benzyl, and—(C₂H₄₀)_(x)H where x has a value from 2 to 5, and X is an anion. Notmore than one of R₂, R₃ or R₄ should be benzyl.

[0292] The preferred alkyl chain length for R₁ is C₁₂-C₁₅, particularlywhere the alkyl group is a mixture of chain lengths derived from coconutor palm kernel fat or is derived synthetically by olefin build up or OXOalcohols synthesis.

[0293] Preferred groups for R₂R₃ and R₄ are methyl and hydroxyethylgroups and the anion X may be selected from halide, methosulfate,acetate and phosphate ions.

[0294] Examples of suitable quaternary ammonium compounds of formulae(i) for use herein are:

[0295] coconut trimethyl ammonium chloride or bromide;

[0296] coconut methyl dihydroxyethyl ammonium chloride or bromide;

[0297] decyl triethyl ammonium chloride;

[0298] decyl dimethyl hydroxyethyl ammonium chloride or bromide;

[0299] C₁₂₋₁₅ dimethyl hydroxyethyl ammonium chloride or bromide;

[0300] coconut dimethyl hydroxyethyl ammonium chloride or bromide;

[0301] myristyl trimethyl ammonium methyl sulfate;

[0302] lauryl dimethyl benzyl ammonium chloride or bromide;

[0303] lauryl dimethyl (ethenoxy)₄ ammonium chloride or bromide;

[0304] choline esters (compounds of formula (i) wherein R₁ is

[0305] alkyl and R₂R₃R₄ are methyl). di-alkyl imidazolines [compounds offormula (i)].

[0306] Other cationic surfactants useful herein are also described inU.S. Pat. No. 4,228,044 and in EP 000 224.

[0307] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 25%, preferablyfrom about 1% to about 8% by weight of such cationic surfactants.

[0308] Ampholytic surfactants are also suitable for use in the laundrydetergent compositions of the present invention. These surfactants canbe broadly described as aliphatic derivatives of secondary or tertiaryamines, or aliphatic derivatives of heterocyclic secondary and tertiaryamines in which the aliphatic radical can be straight- orbranched-chain. One of the aliphatic substituents contains at leastabout 8 carbon atoms, typically from about 8 to about 18 carbon atoms,and at least one contains an anionic water-solubilizing group, e.g.carboxy, sulfonate, sulfate. See U.S. Pat. No. 3,929,678 (column 19,lines 18-35) for examples of ampholytic surfactants.

[0309] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 15%, preferablyfrom about 1% to about 10% by weight of such ampholytic surfactants.

[0310] Zwitterionic surfactants are also suitable for use in laundrydetergent compositions. These surfactants can be broadly described asderivatives of secondary and tertiary amines, derivatives ofheterocyclic secondary and tertiary amines, or derivatives of quaternaryammonium, quaternary phosphonium or tertiary sulfonium compounds. SeeU.S. Pat. No. 3,929,678 (column 19, line 38 through column 22, line 48)for examples of zwitterionic surfactants.

[0311] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 15%, preferablyfrom about 1% to about 10% by weight of such zwitterionic surfactants.

[0312] Semi-polar nonionic surfactants are a special category ofnonionic surfactants which include water-soluble amine oxides containingone alkyl moiety of from about 10 to about 18 carbon atoms and 2moieties selected from the group consisting of alkyl groups andhydroxyalkyl groups containing from about 1 to about 3 carbon atoms;water-soluble phosphine oxides containing one alkyl moiety of from about10 to about 18 carbon atoms and 2 moieties selected from the groupconsisting of alkyl groups and hydroxyalkyl groups containing from about1 to about 3 carbon atoms; and water-soluble sulfoxides containing onealkyl moiety from about 10 to about 18 carbon atoms and a moietyselected from the group consisting of alkyl and hydroxyalkyl moieties offrom about 1 to about 3 carbon atoms.

[0313] Semi-polar nonionic detergent surfactants include the amine oxidesurfactants having the formula:

[0314] wherein R³ is an alkyl, hydroxyalkyl, or alkyl phenyl group ormixtures thereof containing from about 8 to about 22 carbon atoms; R⁴ isan alkylene or hydroxyalkylene group containing from about 2 to about 3carbon atoms or mixtures thereof; x is from 0 to about 3: and each R⁵ isan alkyl or hydroxyalkyl group containing from about 1 to about 3 carbonatoms or a polyethylene oxide group containing from about 1 to about 3ethylene oxide groups. The R⁵ groups can be attached to each other,e.g., through an oxygen or nitrogen atom, to form a ring structure.

[0315] These amine oxide surfactants in particular include C₁₀-C₁₈ alkyldimethyl amine oxides and C₈-C₁₂ alkoxy ethyl dihydroxy ethyl amineoxides.

[0316] When included therein, the laundry detergent compositions of thepresent invention typically comprise from 0.2% to about 15%, preferablyfrom about 1% to about 10% by weight of such semi-polar nonionicsurfactants.

[0317] Builder System

[0318] The compositions according to the present invention may furthercomprise a builder system. Any conventional builder system is suitablefor use herein including aluminosilicate materials, silicates,polycarboxylates and fatty acids, materials such as ethylenediaminetetraacetate, metal ion sequestrants such as aminopolyphosphonates,particularly ethylenediamine tetramethylene phosphonic acid anddiethylene triamine pentamethylenephosphonic acid. Though less preferredfor obvious environmental reasons, phosphate builders can also be usedherein.

[0319] Suitable builders can be an inorganic ion exchange material,commonly an inorganic hydrated aluminosilicate material, moreparticularly a hydrated synthetic zeolite such as hydrated zeolite A, X,B, HS or MAP.

[0320] Another suitable inorganic builder material is layered silicate,e.g. SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consistingof sodium silicate (Na₂Si₂O₅).

[0321] Suitable polycarboxylates containing one carboxy group includelactic acid, glycolic acid and ether derivatives thereof as disclosed inBelgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylatescontaining two carboxy groups include the water-soluble salts ofsuccinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid,diglycollic acid, tartaric acid, tartronic acid and fumaric acid, aswell as the ether carboxylates described in German Offenlegungsschrift2,446,686, and 2,446,487, U.S. Pat. No. 3,935,257 and the sulfinylcarboxylates described in Belgian Patent No. 840,623. Polycarboxylatescontaining three carboxy groups include, in particular, water-solublecitrates, aconitrates and citraconates as well as succinate derivativessuch as the carboxymethyloxysuccinates described in British Patent No.1,379,241, lactoxysuccinates described in Netherlands Application7205873, and the oxypolycarboxylate materials such as2-oxa-1,1,3-propane tricarboxylates described in British Patent No.1,387,447.

[0322] Polycarboxylates containing four carboxy groups includeoxydisuccinates disclosed in British Patent No. 1,261,829,1,1,2,2,-ethane tetracarboxylates, 1,1,3,3-propane tetracarboxylatescontaining sulfo substituents include the sulfosuccinate derivativesdisclosed in British Patent Nos. 1,398,421 and 1,398,422 and in U.S.Pat. No. 3,936,448, and the sulfonated pyrolysed citrates described inBritish Patent No. 1,082,179, while polycarboxylates containingphosphone substituents are is disclosed in British Patent No. 1,439,000.

[0323] Alicyclic and heterocyclic polycarboxylates includecyclopentane-cis,cis-cis-tetracarboxylates, cyclopentadienidepentacarboxylates,2,3,4,5-tetrahydro-furan-cis,cis,cis-tetracarboxylates,2,5-tetrahydro-furan-cis-discarboxylates,2,2,5,5-tetrahydrofuran-tetracarboxylates,1,2,3,4,5,6-hexane-hexacarboxylates and carboxymethyl derivatives ofpolyhydric alcohols such as sorbitol, mannitol and xylitol. Aromaticpolycarboxylates include mellitic acid, pyromellitic acid and thephthalic acid derivatives disclosed in British Patent No. 1,425,343.

[0324] Of the above, the preferred polycarboxylates arehydroxy-carboxylates containing up to three carboxy groups per molecule,more particularly citrates.

[0325] Preferred builder systems for use in the present compositionsinclude a mixture of a water-insoluble aluminosilicate builder such aszeolite A or of a layered silicate (SKS-6), and a water-solublecarboxylate chelating agent such as citric acid.

[0326] A suitable chelant for inclusion in the detergent compositions inaccordance with the invention is ethylenediamine-N,N′-disuccinic acid(EDDS) or the alkali metal, alkaline earth metal, ammonium, orsubstituted ammonium salts thereof, or mixtures thereof. Preferred EDDScompounds are the free acid form and the sodium or magnesium saltthereof. Examples of such preferred sodium salts of EDDS include Na₂EDDSand Na₄EDDS. Examples of such preferred magnesium salts of EDDS includeMgEDDS and Mg₂EDDS. The magnesium salts are the most preferred forinclusion in compositions in accordance with the invention.

[0327] Preferred builder systems include a mixture of a water-insolublealuminosilicate builder such as zeolite A, and a water solublecarboxylate chelating agent such as citric acid.

[0328] Other builder materials that can form part of the builder systemfor use in granular compositions include inorganic materials such asalkali metal carbonates, bicarbonates, silicates, and organic materialssuch as the organic phosphonates, amino polyalkylene phosphonates andamino polycarboxylates.

[0329] Other suitable water-soluble organic salts are the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated form each other bynot more than two carbon atoms.

[0330] Polymers of this type are disclosed in GB-A-1,596,756. Examplesof such salts are polyacrylates of MW 2000-5000 and their copolymerswith maleic anhydride, such copolymers having a molecular weight of from20,000 to 70,000, especially about 40,000.

[0331] Detergency builder salts are normally included in amounts of from5% to 80% by weight of the composition. Preferred levels of builder forliquid detergents are from 5% to 30%.

[0332] Enzymes

[0333] Preferred detergent compositions, in addition to the enzymepreparation of the invention, comprise other enzyme(s) which providescleaning performance and/or fabric care benefits.

[0334] Such enzymes include other proteases, lipases, cutinases,amylases, cellulases, peroxidases, oxidases (e.g. laccases).

[0335] Proteases: Any other protease suitable for use in alkalinesolutions can be used. Suitable proteases include those of animal,vegetable or microbial origin. Microbial origin is preferred. Chemicallyor genetically modified mutants are included. The protease may be aserine protease, preferably an alkaline microbial protease or atrypsin-like protease. Examples of alkaline proteases are subtilisins,especially those derived from Bacillus, e.g., subtilisin Novo,subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168(described in WO 89/06279). Examples of trypsin-like proteases aretrypsin (e.g. of porcine or bovine origin) and the Fusarium proteasedescribed in WO 89/06270.

[0336] Preferred commercially available protease enzymes include thosesold under the trade names Alcalase, Savinase, Primase, Durazym, andEsperase by Novo Nordisk A/S (Denmark), those sold under the trade namesMaxatase, Maxacal, Maxapem, Properase, Purafect and Purafect OXP byGenencor International, and those sold under the trade names Opticleanand Optimase by Solvay Enzymes. Protease enzymes may be incorporatedinto the compositions in accordance with the invention at a level offrom 0.00001% to 2% of enzyme protein by weight of the composition,preferably at a level of from 0.0001% to 1% of enzyme protein by weightof the composition, more preferably at a level of from 0.001% to 0.5% ofenzyme protein by weight of the composition, even more preferably at alevel of from 0.01% to 0.2% of enzyme protein by weight of thecomposition.

[0337] Lipases: Any lipase suitable for use in alkaline solutions can beused. Suitable lipases include those of bacterial or fungal origin.Chemically or genetically modified mutants are included.

[0338] Examples of useful lipases include a Humicola lanuginosa lipase,e.g., as described in EP 258 068 and EP 305 216, a Rhizomucor mieheilipase, e.g., as described in EP 238 023, a Candida lipase, such as a C.antarctica lipase, e.g., the C. antarctica lipase-A or B described in EP214 761, a Pseudomonas lipase such as a P. alcaligenes and P.pseudoalcaligenes lipase, e.g., as described in EP 218 272, a P. cepacialipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., asdisclosed in GB 1,372,034, a P. fluorescens lipase, a Bacillus lipase,e.g., a B. subtilis lipase (Dartois et al., (1993), Biochemica etBiophysica acta 1131, 253-260), a B. stearothermophilus lipase (JP64/744992) and a B. pumilus lipase (WO 91/16422).

[0339] Furthermore, a number. of cloned lipases may be useful, includingthe Penicillium camembertii lipase described by Yamaguchi et al.,(1991), Gene 103, 61-67), the Geotricum candidum lipase (Schimada, Y. etal., (1989), J. Biochem., 106, 383-388), and various Rhizopus lipasessuch as a R. delemar lipase (Hass, M. J et al., (1991), Gene 109,117-113), a R. niveus lipase (Kugimiya et al., (1992), Biosci. Biotech.Biochem. 56, 716-719) and an R. oryzae lipase.

[0340] Other types of lipolytic enzymes such as cutinases may also beuseful, e.g., a cutinase derived from Pseudomonas mendocina as describedin WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g.described in WO 90/09446).

[0341] Especially suitable lipases are lipases such as M1 Lipase™, Lumafast™ and Lipomax™ (Genencor), Lipolase™ and Lipolase Ultra™ (NovoNordisk A/S), and Lipase P “Amano” (Amano Pharmaceutical Co. Ltd.).

[0342] The lipases are normally incorporated in the detergentcomposition at a level of from 0.00001% to 2% of enzyme protein byweight of the composition, preferably at a level of from 0.0001% to 1%of enzyme protein by weight of the composition, more preferably at alevel of from 0.001% to 0.5% of enzyme protein by weight of thecomposition, even more preferably at a level of from 0.01% to 0.2% ofenzyme protein by weight of the composition.

[0343] Amylases: Any amylase (alpha and/or beta) suitable for use inalkaline solutions can be used. Suitable amylases include those ofbacterial or fungal origin. Chemically or genetically modified mutantsare included. Amylases include, for example, alpha-amylases obtainedfrom a special strain of B. licheniformis, described in more detail inGB 1,296,839. Commercially available amylases are Duramyl™, Termamyl™,Fungamyl™ and BAN™ (available from Novo Nordisk A/S) and Rapidase™ andMaxamyl P™ (available from Genencor).

[0344] The amylases are normally incorporated in the detergentcomposition at a level of from 0.00001% to 2% of enzyme protein byweight of the composition, preferably at a level of from 0.0001% to 1%of enzyme protein by weight of the composition, more preferably at alevel of from 0.001% to 0.5% of enzyme protein by weight of thecomposition, even more preferably at a level of from 0.01% to 0.2% ofenzyme protein by weight of the composition.

[0345] Cellulases: Any cellulase suitable for use in alkaline solutionscan be used. Suitable cellulases include those of bacterial or fungalorigin. Chemically or genetically modified mutants are included.Suitable cellulases are disclosed in U.S. Pat. No. 4,435,307, whichdiscloses fungal cellulases produced from Humicola insolens. Especiallysuitable cellulases are the cellulases having color care benefits.Examples of such cellulases are cellulases described in European patentapplication No. 0 495 257.

[0346] Commercially available cellulases include Celluzyme™ produced bya strain of Humicola insolens, (Novo Nordisk A/S), and KAC-500(B)™(KaoCorporation).

[0347] Cellulases are normally incorporated in the detergent compositionat a level of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

[0348] Peroxidases/Oxidases: Peroxidase enzymes are used in combinationwith hydrogen peroxide or a source thereof (e.g. a percarbonate,perborate or persulfate). Oxidase enzymes are used in combination withoxygen. Both types of enzymes are used for “solution bleaching”, i.e. toprevent transfer of a textile dye from a dyed fabric to another fabricwhen said fabrics are washed together in a wash liquor, preferablytogether with an enhancing agent as described in e.g. WO 94/12621 and WO95/01426. Suitable peroxidases/oxidases include those of plant,bacterial or fungal origin. Chemically or genetically modified mutantsare included.

[0349] Peroxidase and/or oxidase enzymes are normally incorporated inthe detergent composition at a level of from 0.00001% to 2% of enzymeprotein by weight of the composition, preferably at a level of from0.0001% to 1% of enzyme protein by weight of the composition, morepreferably at a level of from 0.001% to 0.5% of enzyme protein by weightof the composition, even more preferably at a level of from 0.01% to0.2% of enzyme protein by weight of the composition.

[0350] Mixtures of the above mentioned enzymes are encompassed herein,in particular a mixture of a protease, an amylase, a lipase and/or acellulase.

[0351] The enzyme of the invention, or any other enzyme incorporated inthe detergent composition, is normally incorporated in the detergentcomposition at a level from 0.00001% to 2% of enzyme protein by weightof the composition, preferably at a level from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level from 0.01% to 0.2% of enzyme protein by weight ofthe composition.

[0352] Bleaching Agents

[0353] Additional optional detergent ingredients that can be included inthe detergent compositions of the present invention include bleachingagents such as PB1, PB4 and percarbonate with a particle size of 400-800microns. These bleaching agent components can include one or more oxygenbleaching agents and, depending upon the bleaching agent chosen, one ormore bleach activators. When present, oxygen bleaching compounds willtypically be present at levels of from about 1% to about 25%. Ingeneral, bleaching compounds are optional added components in non-liquidformulations, e.g. granular detergents.

[0354] The bleaching agent component for use herein can be any of thebleaching agents useful for detergent compositions including oxygenbleaches as well as others known in the art.

[0355] The bleaching agent suitable for the present invention can be anactivated or non-activated bleaching agent.

[0356] One category of oxygen bleaching agent that can be usedencompasses percarboxylic acid bleaching agents and salts thereof.Suitable examples of this class of agents include magnesiummonoperoxyphthalate hexahydrate, the magnesium salt of meta-chloroperbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid anddiperoxydodecanedioic acid. Such bleaching agents are disclosed in U.S.Pat. Nos. 4,483,781, 740,446, EP 0 133 354 and U.S. Pat. No. 4,412,934.Highly preferred bleaching agents also include6-nonylamino-6-oxoperoxycaproic acid as described in U.S. Pat. No.4,634,551.

[0357] Another category of bleaching agents that can be used encompassesthe halogen bleaching agents. Examples of hypohalite bleaching agents,for example, include trichloro isocyanuric acid and the sodium andpotassium dichloro isocyanurates and N-chloro and N-bromo alkanesulfonamides. Such materials are normally added at 0.5-10% by weight ofthe finished product, preferably 1-5% by weight.

[0358] The hydrogen peroxide releasing agents can be used in combinationwith bleach activators such as tetra-acetylethylenediamine (TAED),nonanoyloxybenzenesulfonate (NOBS, described in U.S. Pat. No.4,412,934), 3,5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS,described in EP 120 591) or pentaacetylglucose (PAG), which areperhydrolyzed to form a peracid as the active bleaching species, leadingto improved bleaching effect. In addition, very suitable are the bleachactivators C8 (6-octanamido-caproyl) oxybenzene-sulfonate, C9(6-nonanamido caproyl) oxybenzenesulfonate and C10 (6-decanamidocaproyl) oxybenzenesulfonate or mixtures thereof. Also suitableactivators are acylated citrate esters such as disclosed in EuropeanPatent Application No. 91870207.7.

[0359] Useful bleaching agents, including peroxyacids and bleachingsystems comprising bleach activators and peroxygen bleaching compoundsfor use-in cleaning compositions according to the invention aredescribed in application U.S. Ser. No. 08/136,626.

[0360] The hydrogen peroxide may also be present by adding an enzymaticsystem (i.e. an enzyme and a substrate therefore) which is capable ofgeneration of hydrogen peroxide at the beginning or during the washingand/or rinsing process. Such enzymatic systems are disclosed in EuropeanPatent Application EP 0 537 381.

[0361] Bleaching agents other than oxygen bleaching agents are alsoknown in the art and can be utilized herein. One type of non-oxygenbleaching agent of particular interest includes photoactivated bleachingagents such as the sulfonated zinc and/or aluminum phthalocyanines.These materials can be deposited upon the substrate during the washingprocess. Upon irradiation with light, in the presence of oxygen, such asby hanging clothes out to dry in the daylight, the sulfonated zincphthalocyanine is activated and, consequently, the substrate isbleached. Preferred zinc phthalocyanine and a photoactivated bleachingprocess are described in U.S. Pat. No. 4,033,718. Typically, detergentcomposition will contain about 0.025% to about 1.25%, by weight, ofsulfonated zinc phthalocyanine.

[0362] Bleaching agents may also comprise a manganese catalyst. Themanganese catalyst may, e.g., be one of the compounds described in“Efficient manganese catalysts for low-temperature bleaching”, Nature369, 1994, pp. 637-639.

[0363] Suds Suppressors

[0364] Another optional ingredient is a suds suppressor, exemplified bysilicones, and silica-silicone mixtures. Silicones can generally berepresented by alkylated polysiloxane materials, while silica isnormally used in finely divided forms exemplified by silica aerogels andxerogels and hydrophobic silicas of various types. Theses materials canbe incorporated as particulates, in which the suds suppressor isadvantageously releasably incorporated in a water-soluble orwater-dispersible, substantially non surface-active detergentimpermeable carrier. Alternatively the suds suppressor can be dissolvedor dispersed in a liquid carrier and applied by spraying on to one ormore of the other components.

[0365] A preferred silicone suds controlling agent is disclosed in U.S.Pat. No. 3,933,672. Other particularly useful suds suppressors are theself-emulsifying silicone suds suppressors, described in German PatentApplication DTOS 2,646,126. An example of such a-compound is DC-544,commercially available form Dow Corning, which is a siloxane-glycolcopolymer. Especially preferred suds controlling agent are the sudssuppressor system comprising a mixture of silicone oils and2-alkyl-alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanol which iscommercially available under the trade name Isofol 12 R.

[0366] Such suds suppressor system are described in European PatentApplication EP 0 593 841.

[0367] Especially preferred silicone suds controlling agents aredescribed in European Patent Application No. 92201649.8. Saidcompositions can comprise a silicone/silica mixture in combination withfumed nonporous silica such as Aerosil^(R).

[0368] The suds suppressors described above are normally employed atlevels of from 0.001% to 2% by weight of the composition, preferablyfrom 0.01% to 1% by weight.

[0369] Other Components

[0370] Other components used in detergent compositions may be employedsuch as soil-suspending agents, soil-releasing agents, opticalbrighteners, abrasives, bactericides, tarnish inhibitors, coloringagents, and/or encapsulated or nonencapsulated perfumes.

[0371] Especially suitable encapsulating materials are water solublecapsules which consist of a matrix of polysaccharide and polyhydroxycompounds such as described in GB 1,464,616.

[0372] Other suitable water soluble encapsulating materials comprisedextrins derived from ungelatinized starch acid esters of substituteddicarboxylic acids such as described in U.S. Pat. No. 3,455,838. Theseacid-ester dextrins are, preferably, prepared from such starches as waxymaize, waxy sorghum, sago, tapioca and potato. Suitable examples of saidencapsulation materials include N-Lok manufactured by National Starch.The N-Lok encapsulating material consists of a modified maize starch andglucose. The starch is modified by adding mono-functional substitutedgroups such as octenyl succinic acid anhydride.

[0373] Antiredeposition and soil suspension agents suitable hereininclude cellulose derivatives such as methylcellulose,carboxymethylcellulose and hydroxyethylcellulose, and homo- orco-polymeric polycarboxylic acids or their salts. Polymers of this typeinclude the polyacrylates and maleic anhydride-acrylic acid copolymerspreviously mentioned as builders, as well as copolymers of maleicanhydride with ethylene, methylvinyl ether or methacrylic acid, themaleic anhydride constituting at least 20 mole percent of the copolymer.These materials are normally used at levels of from 0.5% to 10% byweight, more preferably form 0.75% to 8%, most preferably from 1% to 6%by weight of the composition.

[0374] Preferred optical brighteners are anionic in character, examplesof which are disodium4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2:2′-disulfonate,disodium4,4′-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino-stilbene-2:2′-disulfonate,disodium4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2:2′-disulfonate,monosodium4′,4″-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2-sulphonate,disodium4,4′-bis-(2-anilino-4-(N-methyl-N-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulfonate,disodium 4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2′disulfonate, disodium4,4′-bis(2-anilino-4-(1-methyl-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,sodium 2(stilbyl-4″-(naphtho-1′,2′:4,5)-1,2,3-triazole-2″-sulphonate and4,4′-bis(2-sulphostyryl)biphenyl.

[0375] Other useful polymeric materials are the polyethylene glycols,particularly those of molecular weight 1000-10000, more particularly2000 to 8000 and most preferably about 4000. These are used at levels offrom 0.20% to 5% more preferably from 0.25% to 2.5% by weight. Thesepolymers and the previously mentioned homo- or co-polymericpoly-carboxylate salts are valuable for improving whiteness maintenance,fabric ash deposition, and cleaning performance on clay, proteinaceousand oxidizable soils in the presence of transition metal impurities.

[0376] Soil release agents useful in compositions of the presentinvention are conventionally copolymers or terpolymers of terephthalicacid with ethylene glycol and/or propylene glycol units in variousarrangements. Examples of such polymers are disclosed in U.S. Pat. Nos.4,116,885 and 4,711,730 and EP 0 272 033. A particular preferred polymerin accordance with EP 0 272 033 has the formula:

(CH₃(PEG)₄₃)_(0.75)(POH)_(0.25)[T-PO)_(2.8)(T-PEG)_(0.4)]T(POH)_(0.25)((PEG)₄₃CH₃)_(0.75)

[0377] where PEG is —(OC₂H₄)O—, PO is (OC₃H₆O) and T is (pOOC₆H₄CO).

[0378] Also very useful are modified polyesters as random copolymers ofdimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and1,2-propanediol, the end groups consisting primarily of sulfobenzoateand secondarily of mono esters of ethylene glycol and/or1,2-propanediol. The target is to obtain a polymer capped at both end bysulfobenzoate groups, “primarily”, in the present context most of saidcopolymers herein will be end-capped by sulfobenzoate groups. However,some copolymers will be less than fully capped, and therefore their endgroups may consist of monoesters of ethylene glycol and/or1,2-propanediol, thereof consist “secondarily” of such species.

[0379] The selected polyesters herein contain about 46% by weight ofdimethyl terephthalic acid, about 16% by weight of 1,2-propanediol,about 10% by weight ethylene glycol, about 13% by weight of dimethylsulfobenzoic acid and about 15% by weight of sulfoisophthalic acid, andhave a molecular weight of about 3.000. The polyesters and their methodof preparation are described in detail in EP 311 342.

[0380] Softening agents: Fabric softening agents can also beincorporated into laundry detergent compositions in accordance with thepresent invention. These agents may be inorganic or organic in type.Inorganic softening agents are exemplified by the smectite claysdisclosed in GB-A-1 400898 and in U.S. Pat. No. 5,019,292. Organicfabric softening agents include the water insoluble tertiary amines asdisclosed in GB-A1 514 276 and EP 0 011 340 and their combination withmono C₁₂-C₁₄ quaternary ammonium salts are disclosed in EP-B-0 026 528and di-long-chain amides as disclosed in EP 0 242 919. Other usefulorganic ingredients of fabric softening systems include high molecularweight polyethylene oxide materials as disclosed in EP 0 299 575 and 0313 146.

[0381] Levels of smectite clay are normally in the range from 5% to 15%,more preferably from 8% to 12% by weight, with the material being addedas a dry mixed component to the remainder of the formulation. Organicfabric softening agents such as the water-insoluble tertiary amines ordi-long chain amide materials are incorporated at levels of from 0.5% to5% by weight, normally from 1% to 3% by weight whilst the high molecularweight polyethylene oxide materials and the water soluble cationicmaterials are added at levels of from 0.1% to 2%, normally from 0.15% to1.5% by weight. These materials are normally added to the spray driedportion of the composition, although in some instances it may be moreconvenient to add them as a dry mixed particulate, or spray them asmolten liquid on to other solid components of the composition.

[0382] Polymeric dye-transfer inhibiting agents: The detergentcompositions according to the present invention may also comprise from0.001% to 10%, preferably from 0.01% to 2%, more preferably form 0.05%to 1% by weight of polymeric dye-transfer inhibiting agents. Saidpolymeric dye-transfer inhibiting agents are normally incorporated intodetergent compositions in order to inhibit the transfer of dyes fromcolored fabrics onto fabrics washed therewith. These polymers have theability of complexing or adsorbing the fugitive dyes washed out of dyedfabrics before the dyes have the opportunity to become attached to otherarticles in the wash.

[0383] Especially suitable polymeric dye-transfer inhibiting agents arepolyamine N-oxide polymers, copolymers of N-vinyl-pyrrolidone andN-vinylimidazole, polyvinylpyrrolidone polymers, polyvinyloxazolidonesand polyvinylimidazoles or mixtures thereof.

[0384] Addition of such polymers also enhances the performance of theenzymes according the invention.

[0385] The detergent composition according to the invention can be inliquid, paste, gels, bars or granular forms.

[0386] Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. No. 4,106,991 and 4,661,452 (both to Novo Industri A/S) andmay optionally be coated by methods known in the art. Examples of waxycoating materials are poly(ethylene oxide) products (polyethyleneglycol,PEG) with mean molecular weights of 1000 to 20000; ethoxylatednonylphenols having from 16 to 50 ethylene oxide units; ethoxylatedfatty alcohols in which the alcohol contains from 12 to 20 carbon atomsand in which there are 15 to 80 ethylene oxide units; fatty alcohols;fatty acids; and mono- and di- and triglycerides of fatty acids.Examples of film-forming coating materials suitable for application byfluid bed techniques are given in GB 1483591.

[0387] Granular compositions according to the present invention can alsobe in “compact form”, i.e. they may have a relatively higher densitythan conventional granular detergents, i.e. form 550 to 950 g/l; in suchcase, the granular detergent compositions according to the presentinvention will contain a lower amount of “Inorganic filler salt”,compared to conventional granular detergents; typical filler salts arealkaline earth metal salts of sulfates and chlorides, typically sodiumsulfate; “Compact” detergent typically comprise not more than 10% fillersalt. The liquid compositions according to the present invention canalso be in “concentrated form”, in such case, the liquid detergentcompositions according to the present invention will contain a loweramount of water, compared to conventional liquid detergents. Typically,the water content of the concentrated liquid detergent is less than 30%,more preferably less than 20%, most preferably less than 10% by weightof the detergent compositions.

[0388] The compositions of the invention may for example, be formulatedas hand and machine laundry detergent compositions including laundryadditive compositions and compositions suitable for use in thepretreatment of stained fabrics, rinse added fabric softenercompositions, and compositions for use in general household hard surfacecleaning operations and dishwashing operations.

[0389] The following examples are meant to exemplify compositions forthe present invention, but are not necessarily meant to limit orotherwise define the scope of the invention.

[0390] In the detergent compositions, the abbreviated componentidentifications have the following meanings: LAS: Sodium linear C₁₂alkyl benzene sulfonate TAS: Sodium tallow alkyl sulfate XYAS: SodiumC_(1X)-C_(1Y) alkyl sulfate SS: Secondary soap surfactant of formula2-butyl octanoic acid 25EY: A C₁₂-C₁₅ predominantly linear primaryalcohol condensed with an average of Y moles of ethylene oxide 45EY: AC₁₄-C₁₅ predominantly linear primary alcohol condensed with an averageof Y moles of ethylene oxide XYEZS: C_(1X)-C_(1Y) sodium alkyl sulfatecondensed with an average of Z moles of ethylene oxide per moleNonionic: C₁₃-C₁₅ mixed ethoxylated/propoxylated fatty alcohol with anaverage degree of ethoxylation of 3.8 and an average degree ofpropoxylation of 4.5 sold under the trade name Plurafax LF404 by BASFGmbH CFAA: C₁₂-C₁₄ alkyl N-methyl glucamide TFAA: C₁₆-C₁₈ alkyl N-methylglucamide Silicate: Amorphous Sodium Silicate (SiO₂:Na₂O ratio = 2.0)NaSKS-6: Crystalline layered silicate of formula δ-Na₂Si₂O₅ Carbonate:Anhydrous sodium carbonate Phosphate: Sodium tripolyphosphate MA/AA:Copolymer of 1:4 maleic/acrylic acid, average molecular weight about80,000 Polyacrylate: Polyacrylate homopolymer with an average molecularweight of 8,000 sold under the trade name PA30 by BASF GmbH Zeolite A:Hydrated Sodium Aluminosilicate of formula Na₁₂(AlO₂SiO₂)₁₂.27H₂O havinga primary particle size in the range from 1 to 10 micrometers Citrate:Tri-sodium citrate dihydrate Citric: Citric Acid Perborate: Anhydroussodium perborate monohydrate bleach, empirical formula NaBO₂.H₂O₂ PB4:Anhydrous sodium perborate tetrahydrate Percarbonate: Anhydrous sodiumpercarbonate bleach of empirical formula 2Na₂CO₃.3H₂O₂ TAED: Tetraacetylethylene diamine CMC: Sodium carboxymethyl cellulose DETPMP: Diethylenetriamine penta (methylene phosphonic acid), marketed by Monsanto underthe Trade name Dequest 2060 PVP: Polyvinylpyrrolidone polymer EDDS:Ethylenediamine-N, N′-disuccinic acid, [S,S] isomer in the form of thesodium salt Suds 25% paraffin wax Mpt 50° C., 17% hydrophobic silica,58% Suppressor: paraffin oil Granular Suds: 12% Silicone/silica, 18%stearyl alcohol, 70% Suppressor: starch in granular form Sulfate:Anhydrous sodium sulfate HMWPEO: High molecular weight polyethyleneoxide TAE 25: Tallow alcohol ethoxylate (25)

DETERGENT EXAMPLE I

[0391] A granular fabric cleaning composition in accordance with theinvention may be prepared as follows: Sodium linear C₁₂ alkyl 6.5benzene sulfonate Sodium sulfate 15.0 Zeolite A 26.0 Sodiumnitrilotriacetate 5.0 Enzyme of the invention 0.1 PVP 0.5 TAED 3.0 Boricacid 4.0 Perborate 18.0 Phenol sulfonate 0.1 Minors Up to 100

DETERGENT EXAMPLE II

[0392] A compact granular fabric cleaning composition (density 800 g/l)in accord with the invention may be prepared as follows: 45AS 8.0 25E3S2.0 25E5 3.0 25E3 3.0 TFAA 2.5 Zeolite A 17.0 NaSKS-6 12.0 Citric acid3.0 Carbonate 7.0 MA/AA 5.0 CMC 0.4 Enzyme of the invention 0.1 TAED 6.0Percarbonate 22.0 EDDS 0.3 Granular suds suppressor 3.5 water/minors Upto 100%

DETERGENT EXAMPLE Ill

[0393] Granular fabric cleaning compositions in accordance with theinvention which are especially useful in the laundering of coloredfabrics were prepared as follows: LAS 10.7  — TAS 2.4 — TFAA — 4.0 45AS3.1 10.0  45E7 4.0 — 25E3S — 3.0 68E11 1.8 — 25E5 — 8.0 Citrate 15.0 7.0 Carbonate — 10   Citric acid 2.5 3.0 Zeolite A 32.1  25.0  Na-SKS-6— 9.0 MA/AA 5.0 5.0 DETPMP 0.2 0.8 Enzyme of the invention  0.10  0.05Silicate 2.5 — Sulfate 5.2 3.0 PVP 0.5 — Poly (4-vinylpyridine)-N- — 0.2Oxide/copolymer of vinyl- imidazole and vinyl- pyrrolidone Perborate 1.0Phenol sulfonate 0.2 Water/Minors Up to 100%

DETERGENT EXAMPLE IV

[0394] Granular fabric cleaning compositions in accordance with theinvention which provide “Softening through the wash” capability may beprepared as follows: 45AS — 10.0  LAS 7.6 — 68AS 1.3 — 45E7 4.0 — 25E3 —5.0 Coco-alkyl-dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride Citrate5.0 3.0 Na-SKS-6 — 11.0  Zeolite A 15.0  15.0  MA/AA 4.0 4.0 DETPMP 0.40.4 Perborate 15.0  — Percarbonate — 15.0  TAED 5.0 5.0 Smectite clay10.0  10.0  HMWPEO — 0.1 Enzyme of the invention  0.10  0.05 Silicate3.0 5.0 Carbonate 10.0  10.0  Granular suds suppressor 1.0 4.0 CMC 0.20.1 Water/Minors Up to 100%

DETERGENT EXAMPLE V

[0395] Heavy duty liquid fabric cleaning compositions in accordance withthe invention may be prepared as follows: I II LAS acid form — 25.0 Citric acid 5.0 2.0 25AS acid form 8.0 — 25AE2S acid form 3.0 — 25AE78.0 — CFAA 5   — DETPMP 1.0 1.0 Fatty acid 8   — Oleic acid — 1.0Ethanol 4.0 6.0 Propanediol 2.0 6.0 Enzyme of the invention  0.10  0.05Coco-alkyl dimethyl — 3.0 hydroxy ethyl ammonium chloride Smectite clay— 5.0 PVP 2.0 — Water/Minors Up to 100%

[0396] Leather Industry Applications

[0397] A subtilase of the invention may be used in the leather industry,in particular for use in depilation of skins.

[0398] In said application a subtilase variant of the invention ispreferably used in an enzyme composition which further comprises anotherprotease.

[0399] For a more detailed description of suitable other proteases seesection relating to suitable enzymes for use in a detergent composition(vide supra).

[0400] Wool Industry Applications

[0401] A subtilase of the invention may be used in the wool industry, inparticular for use in cleaning of clothes comprising wool.

[0402] In said application a subtilase variant of the invention ispreferably used in an enzyme composition which further comprises anotherprotease.

[0403] For a more detailed description of suitable other proteases seesection relating to suitable is enzymes for use in a detergentcomposition (vide supra).

[0404] The invention is described in further detail in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed.

[0405] Materials and Methods

[0406] Strains:

[0407]B. subtilis DN1885 (Diderichsen et al., 1990).

[0408]B. lentus 309 and 147 are specific strains of Bacillus lentus,deposited with the NCIB and accorded the accession numbers NCIB 10309and 10147, and described in U.S. Pat. No. 3,723,250 incorporated byreference herein.

[0409]E. MC 1000 (M. J. Casadaban and S. N. Cohen (1980); J. Mol. Biol.138 179-207), was made r⁻,m⁺ by conventional methods and is alsodescribed in U.S. patent application Ser. No. 039,298.

[0410] Plasmids:

[0411] pJS3: E. coli—B. subtilis shuttle vector containing a syntheticgene encoding for subtilase 309. (Described by Jacob Schiødt et al. inProtein and Peptide letters 3:39-44 (1996)).

[0412] pSX222: B. subtilis expression vector (Described in WO 96/34946).General Molecular Biology Methods:

[0413] Unless otherwise mentioned the DNA manipulations andtransformations were performed using standard methods of molecularbiology (Sambrook et al. (1989) Molecular cloning: A laboratory manual,Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.) “Current protocols in Molecular Biology”. John Wiley and Sons,1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular BiologicalMethods for Bacillus”. John Wiley and Sons, 1990).

[0414] Enzymes for DNA manipulations were used according to thespecifications of the suppliers.

[0415] Enzymes for DNA Manipulations

[0416] Unless otherwise mentioned all enzymes for DNA manipulations,such as e.g. restriction endonucleases, ligases etc., are obtained fromNew England Bolas, Inc.

[0417] Proteolytic Activity

[0418] In the context of this invention proteolytic activity isexpressed in Kilo NOVO Protease Units (KNPU). The activity is determinedrelatively to an enzyme standard (SAVINASE®), and the determination isbased on the digestion of a dimethyl casein (DMC) solution by theproteolytic enzyme at standard conditions, i.e. 50° C., pH 8.3, 9 min.reaction time, 3 min. measuring time. A folder AF 220/1 is availableupon request to Novo Nordisk A/S, Denmark, which folder is herebyincluded by reference.

[0419] A GU is a Glycine Unit, defined as the proteolytic enzymeactivity which, under standard conditions, during a 15 minute incubationat 40° C., with N-acetyl casein as substrate, produces an amount ofNH₂-group equivalent to 1 mmole of glycine.

[0420] Enzyme activity can also be measured using the PNA assay,according to reaction with the soluble substratesuccinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, whichis described in the Journal of American Oil Chemists Society, Rothgeb,T. M., Goodlander, B. D., Garrison, P. H., and Smith, L. A., (1988).

[0421] Fermentation:

[0422] Fermentation of subtilase enzymes were performed at 30° C. on arotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flaskscontaining 100 ml BPX medium for 5 days.

[0423] Consequently in order to produce e.g. 2 liter broth, 20Erlenmeyer flasks were fermented simultaneously.

[0424] Media: BPX: Composition (per liter) Potato starch 100 g Groundbarley  50 g Soybean flour  20 g Na₂HPO₄X12H₂O  9 g Pluronic  0.1 gSodium caseinate  10 g

[0425] The starch in the medium is liquefied with alpha-amylase and themedium is sterilized by heating at 120° C. for 45 minutes. Aftersterilization the pH of the medium is adjusted to 9 by addition ofNaHCO₃ to 0.1 M.

EXAMPLE1

[0426] Construction and Expression of Enzyme Variants

[0427] Site-Directed Mutagenesis:

[0428] Subtilase 309 site-directed variants, where specific insertionswere performed in one the active site loops according to the invention,was made by the “Unique site elimination (USE)” or the “Uracil-USE”technique described respectively by Deng et al. (Anal. Biochem.200:81-88 (1992)) and Markvardsen et al. (BioTechniques 18(3):371-372(1995)).

[0429] The template plasmid was pJS3, or an analogue of this containinga variant of subtilase 309, e.g. USE mutagenesis was performed with anoligonucleotide directed to the construction of a G97GASG insertionvariant resulting in a final G97GASG Subtilase 309 variant.

[0430] The subtilase 309 variants constructed in pJS3 were thensubcloned into the B. subtilis pSX222 expression plasmid, using therestriction enzymes KpnI and MluI.

[0431] Localized Random Mutagenesis in Order to Insert Random Insertionsin a Localized Region:

[0432] The overall strategy used to perform localized random mutagenesiswas:

[0433] a mutagenic primer (oligonucleotide) was synthesized whichcorresponds to the part of the DNA sequence to be modified except forthe nucleotide(s) corresponding to amino acid codon(s) to be modified byinsertions.

[0434] Subsequently, the resulting mutagenic primer was used in a PCRreaction with a suitable opposite primer. The resulting PCR fragment waspurified and digested and cloned into a E coli-B. subtilis shuttlevector.

[0435] Alternatively, and if necessary, the resulting PCR fragment isused in a second PCR reaction as a primer with a second suitableopposite primer so as to allow digestion and cloning of the mutagenizedregion into the shuttle vector. The PCR reactions are performed undernormal conditions.

[0436] Following this strategy a localized random library wasconstructed in SAVINASE wherein insertions were introduced in the activesite loop region from 95-103.

[0437] The mutations/insertions were introduced by mutagenic primers (sebelow), so that only four amino acids: Thr, Gly, Ala and Ser arerepresented with two codons each (R=50% A and G; S=50% C and G; andY=50% C and T). The produced PCR fragment were cloned into the Avr IIand Not I sites of plasmid pJS3, and ten randomly chosen E. colicolonies were sequenced to confirm the mutations designed.

[0438] The mutagenic primer (5′-CTA TAC GCT AAA GTC CTA GGG GCG RSY RSYRSY RSY RSY RSY RSY GTC AGC TCG ATT GCC CM GG-3′ (sense) (SEQ ID NO:17)) were used in a PCR reaction with a suitable anti-sense oppositeprimer, situated downstream of the MluI site in pJS3 (e.g. 5′-CCC TTTAAC CGC ACA GCG TTT-3′ (anti-sense) (SEQ ID NO: 18)) and the plasmidpJS3 as template. This resulting PCR product was cloned into the pJS3shuttle vector by using the restriction enzymes Avr II and Not I.

[0439] The localized random library constructed in pJS3 was thensubcloned into the B. subtilis pSX222 expression plasmid, using therestriction enzymes KpnI and MluI.

[0440] The library prepared contained approximately 100,000 individualclones/library.

[0441] Ten randomly chosen colonies were sequenced to confirm themutations designed.

[0442] In order to purify a subtilase variant of the invention the B.subtilis pSX222 expression plasmid comprising a variant of the inventionwas transformed into a competent B. subtilis strain and was fermented asdescribed above in a medium containing 10 micrograms/ml chloramphenicol(CAM).

EXAMPLE2

[0443] Purification of Enzyme Variants

[0444] This procedure describes the purification of a 2 liter scalefermentation of subtilisin 147, subtilisin 309 or mutants thereof.

[0445] Approximately 1.6 liters of fermentation broth were centrifugedat 5000 rpm for 35 minutes in 1 liter beakers. The supernatants wereadjusted to pH 6.5 using 10% acetic acid and filtered on Seitz SupraS100 filter plates.

[0446] The filtrates were concentrated to approximately 400 ml using anAmicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UFconcentrate was centrifuged and filtered prior to absorption at roomtemperature on a Bacitracin affinity column at pH 7. The protease waseluted from the Bacitracin column at room temperature using 25%2-propanol and 1 M sodium chloride in a buffer solution with 0.01dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chlorideadjusted to pH 7.

[0447] The fractions with protease activity from the Bacitracinpurification step were combined and applied to a 750 ml Sephadex G25column (5 cm dia.) equilibrated with a buffer containing 0.01dimethylglutaric acid, 0.2 M boric acid and 0.002 M calcium chlorideadjusted to pH 6.5.

[0448] Fractions with proteolytic activity from the Sephadex G25 columnwere combined and applied to a 150 ml CM Sepharose CL 6B cation exchangecolumn (5 cm dia.) equilibrated with a buffer containing 0.01 Mdimethylglutaric acid, 0.2 M boric acid, and 0.002 M calcium chlorideadjusted to pH 6.5.

[0449] The protease was eluted using a linear gradient of 0-0.1 M sodiumchloride in 2 liters of the same buffer (0-0.2 M sodium chloride in caseof subtilisin 147).

[0450] In a final purification step protease containing fractions fromthe CM Sepharose column were combined and concentrated in an Amiconultrafiltration cell equipped with a GR81PP membrane (from the DanishSugar Factories Inc.).

[0451] By using the techniques of Example 1 for the construction and theabove isolation procedure the following subtilisin 309 variants wereproduced and isolated:

[0452] 37.03: G97GASG+A98S+S99G+G100A+S101A;

[0453] 37.06: G97GAA+A98S+S99G+S101T; and

[0454]137.04: G97GAS+A98S+S99G.

EXAMPLE 3

[0455] Wash Performance of Detergent Compositions Comprising EnzymeVariants

[0456] The following example provides results from a number of washingtests that were conducted under the conditions indicated

[0457] Experimental Conditions TABLE III Experimental conditions forevaluation of Subtilisin 309 variants. Detergent Protease ModelDetergent 95 Detergent dose 3.0 g/l pH 10.5 Wash time  15 min.Temperature 15° C. Water hardness 6°dH Enzymes Subtilisin 309 variantsas listed below Enzyme conc.  10 nM Test system 150 ml glass beakerswith a stirring rod Textile/volume 5 textile pieces ( 2.5 cm) in 50 mldetergent Test material EMPA117 from Center for Testmaterials, Holland

[0458] The detergent used is a simple model formulation. pH is adjustedto 10.5 which is within the normal range for a powder detergent. Thecomposition of model detergent 95 is as follows: STP (Na₅P₃O₁₀)  25%Na₂SO₄  25% Na₂CO₃  10% LAS (Nansa 80S)  20% Nonionic tenside (Dobanol25-7) 5.0% Na₂Si₂O₅ 5.0% Carboxymethylcellulose (CMC) 0.5% Water 9.5%

[0459] Water hardness was adjusted by adding CaCl₂ and MgCl₂(Ca²⁺:Mg²⁺=2:1) to deionized water (see also Surfactants in ConsumerProducts—Theory, Technology and Application, Springer Verlag 1986). pHof the detergent solution was adjusted to pH 10.5 by addition of HCl.

[0460] Measurement of reflectance (R) on the test material was done at460 nm using a Macbeth ColorEye 7000 photometer. The measurements weredone according to the manufacturer's protocol.

[0461] The wash performance of the Subtilisin 309 variants was evaluatedby calculating a performance factor:

P=(R _(vaniant) −R _(blank))−(R _(Savinase) −R _(blank))

[0462] wherein

[0463] P: Performance factor

[0464] R_(Variant): Reflectance of test material washed with variant

[0465] R_(Savinase): Reflectance of test material washed with Savinase®

[0466] R_(Blank): Reflectance of test material washed with no enzyme

[0467] The claimed Subtilisin 309 variants all have improved washperformance compared to Savinase®—i.e. P>1.

[0468] The variants are divided into improvement classes designated withcapital letters:

[0469] Class A: 1<P≦1.5

[0470] Class B: 1.5<P≦2

[0471] Class C: P>2 TABLE IV Subtilisin 309 variants and improvementclasses. Improvement class Variants A 37.03: G97GASG + A98S + S99G +G100A + S101A 37.04: G97GAS + A98S + S99G B 37.06: G97GAA + A98S +S99G + S101T C

[0472] As it can be seen from Table IV SAVINASE® variants of theinvention exhibits an improvement in wash performance.

1 18 1 4 PRT Artificial Sequence Variation 1 Gly Lys Ala Ser 1 4 2 6 PRTArtificial Sequence Variation 2 Ala Gly Lys Ala Ser Leu 1 5 3 4 PRTArtificial Sequence Variation 3 Ala Gly Gly Leu 1 4 275 PRT Bacillus 4Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu 1 5 1015 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 2530 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 35 4045 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 50 5560 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly 65 7075 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 8590 95 Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu100 105 110 Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu GlyGly 115 120 125 Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys AlaVal Ala 130 135 140 Ser Gly Val Val Val Ala Ala Ala Ala Gly Asn Glu GlyThr Ser Gly 145 150 155 160 Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys TyrPro Ser Val Ile Ala 165 170 175 Val Gly Ala Val Asp Ser Ser Asn Gln ArgAla Ser Phe Ser Ser Val 180 185 190 Gly Pro Glu Leu Asp Val Met Ala ProGly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly Asn Lys Tyr Gly AlaTyr Asn Gly Thr Ser Met Ala Ser 210 215 220 Pro His Val Ala Gly Ala AlaAla Leu Ile Leu Ser Lys His Pro Asn 225 230 235 240 Trp Thr Asn Thr GlnVal Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp SerPhe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln275 5 268 PRT Bacillus 5 Gln Thr Val Pro Trp Gly Ile Ser Phe Ile Asn ThrGln Gln Ala His 1 5 10 15 Asn Arg Gly Ile Phe Gly Asn Gly Ala Arg ValAla Val Leu Asp Thr 20 25 30 Gly Ile Ala Ser His Pro Asp Leu Arg Ile AlaGly Gly Ala Ser Phe 35 40 45 Ile Ser Ser Glu Pro Ser Tyr His Asp Asn AsnGly His Gly Thr His 50 55 60 Val Ala Gly Thr Ile Ala Ala Leu Asn Asn SerIle Gly Val Leu Gly 65 70 75 80 Val Arg Pro Ser Ala Asp Leu Tyr Ala LeuLys Val Leu Asp Arg Asn 85 90 95 Gly Ser Gly Ser Leu Ala Ser Val Ala GlnGly Ile Glu Trp Ala Ile 100 105 110 Asn Asn Asn Met His Ile Ile Asn MetSer Leu Gly Ser Thr Ser Gly 115 120 125 Ser Ser Thr Leu Glu Leu Ala ValAsn Arg Ala Asn Asn Ala Gly Ile 130 135 140 Leu Leu Val Gly Ala Ala GlyAsn Thr Gly Arg Gln Gly Val Asn Tyr 145 150 155 160 Pro Ala Arg Tyr SerGly Val Met Ala Val Ala Ala Val Asp Gln Asn 165 170 175 Gly Gln Arg AlaSer Phe Ser Thr Tyr Gly Pro Glu Ile Glu Ile Ser 180 185 190 Ala Pro GlyVal Asn Val Asn Ser Thr Tyr Thr Gly Asn Arg Tyr Val 195 200 205 Ser LeuSer Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Val Ala 210 215 220 AlaLeu Val Lys Ser Arg Tyr Pro Ser Tyr Thr Asn Asn Gln Ile Arg 225 230 235240 Gln Arg Ile Asn Gln Thr Ala Thr Tyr Leu Gly Ser Pro Ser Leu Tyr 245250 255 Gly Asn Gly Leu Val His Ala Gly Arg Ala Thr Gln 260 265 6 268PRT Bacillus 6 Gln Thr Val Pro Trp Gly Ile Asn Arg Val Gln Ala Pro IleAla Gln 1 5 10 15 Ser Arg Gly Phe Thr Gly Thr Gly Val Arg Val Ala ValLeu Asp Thr 20 25 30 Gly Ile Ser Asn His Ala Asp Leu Arg Ile Arg Gly GlyAla Ser Phe 35 40 45 Val Pro Gly Glu Pro Asn Ile Ser Asp Gly Asn Gly HisGly Thr Gln 50 55 60 Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile GlyVal Leu Gly 65 70 75 80 Val Ala Pro Asn Val Asp Leu Tyr Gly Val Lys ValLeu Gly Ala Ser 85 90 95 Gly Ser Gly Ser Ile Ser Gly Ile Ala Gln Gly LeuGln Trp Ala Ala 100 105 110 Asn Asn Gly Met His Ile Ala Asn Met Ser LeuGly Ser Ser Ala Gly 115 120 125 Ser Ala Thr Met Glu Gln Ala Val Asn GlnAla Thr Ala Ser Gly Val 130 135 140 Leu Val Val Ala Ala Ser Gly Asn SerGly Ala Gly Asn Val Gly Phe 145 150 155 160 Pro Ala Arg Tyr Ala Asn AlaMet Ala Val Gly Ala Thr Asp Gln Asn 165 170 175 Asn Asn Arg Ala Thr PheSer Gln Tyr Gly Ala Gly Leu Asp Ile Val 180 185 190 Ala Pro Gly Val GlyVal Gln Ser Thr Val Pro Gly Asn Gly Tyr Ala 195 200 205 Ser Phe Asn GlyThr Ser Met Ala Thr Pro His Val Ala Gly Val Ala 210 215 220 Ala Leu ValLys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile Arg 225 230 235 240 AsnHis Leu Lys Asn Thr Ala Thr Asn Leu Gly Asn Thr Thr Gln Phe 245 250 255Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265 7 269 PRTBacillus 7 Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro AlaAla 1 5 10 15 His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala ValLeu Asp 20 25 30 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly GlyAla Ser 35 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly HisGly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile GlyVal Leu 65 70 75 80 Gly Val Ala Pro Asn Ala Glu Leu Tyr Ala Val Lys ValLeu Gly Ala 85 90 95 Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly LeuGlu Trp Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser LeuGly Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn SerAla Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn SerGly Ala Gly Ser Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn AlaMet Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser PheSer Gln Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val AsnVal Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn GlyThr Ser Met Ala Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu ValLys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240 Arg AsnHis Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255 TyrGly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265 8 274 PRTBacillus 8 Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp LysVal 1 5 10 15 Gln Ala Gln Gly Tyr Lys Gly Ala Asn Val Lys Val Gly IleIle Asp 20 25 30 Thr Gly Ile Ala Ala Ser Val Glu Ala Ala Ala Gln His ThrAsp Leu 35 40 45 Lys Val Val Gly Gly Ala Ser Phe Val Ser Gly Glu Ser TyrAsn Thr 50 55 60 Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val AlaAla Leu 65 70 75 80 Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Asn ValSer Leu Tyr 85 90 95 Ala Ile Lys Val Leu Asn Ser Ser Gly Ser Gly Thr TyrSer Ala Ile 100 105 110 Val Ser Gly Ile Glu Trp Ala Thr Gln Asn Gly LeuAsp Val Ile Asn 115 120 125 Met Ser Leu Gly Gly Pro Ser Gly Ser Thr AlaLeu Lys Gln Ala Val 130 135 140 Asp Lys Ala Tyr Ala Ser Gly Ile Val ValVal Ala Ala Ala Gly Asn 145 150 155 160 Ser Gly Ser Ser Gly Ser Gln AsnThr Ile Gly Tyr Pro Ala Lys Tyr 165 170 175 Asp Ser Val Ile Ala Val GlyAla Val Asp Ser Asn Lys Asn Arg Ala 180 185 190 Ser Phe Ser Ser Val GlyAla Glu Leu Glu Val Met Ala Pro Gly Val 195 200 205 Ser Val Tyr Ser ThrTyr Pro Ser Asn Thr Tyr Thr Ser Leu Asn Gly 210 215 220 Thr Ser Met AlaSer Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu 225 230 235 240 Ser LysTyr Pro Thr Leu Ser Ala Ser Gln Val Arg Asn Arg Leu Ser 245 250 255 SerThr Ala Thr Asn Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu 260 265 270Ile Asn 9 279 PRT Bacillus 9 Tyr Thr Pro Asn Asp Pro Tyr Phe Ser Ser ArgGln Tyr Gly Pro Gln 1 5 10 15 Lys Ile Gln Ala Pro Gln Ala Trp Asp IleAla Glu Gly Ser Gly Ala 20 25 30 Lys Ile Ala Ile Val Asp Thr Gly Val GlnSer Asn His Pro Asp Leu 35 40 45 Ala Gly Lys Val Val Gly Gly Trp Asp PheVal Asp Asn Asp Ser Thr 50 55 60 Pro Gln Asn Gly Asn Gly His Gly Thr HisCys Ala Gly Ile Ala Ala 65 70 75 80 Ala Val Thr Asn Asn Ser Thr Gly IleAla Gly Thr Ala Pro Lys Ala 85 90 95 Ser Ile Leu Ala Val Arg Val Leu AspAsn Ser Gly Ser Gly Thr Trp 100 105 110 Thr Ala Val Ala Asn Gly Ile ThrTyr Ala Ala Asp Gln Gly Ala Lys 115 120 125 Val Ile Ser Leu Ser Leu GlyGly Thr Val Gly Asn Ser Gly Leu Gln 130 135 140 Gln Ala Val Asn Tyr AlaTrp Asn Lys Gly Ser Val Val Val Ala Ala 145 150 155 160 Ala Gly Asn AlaGly Asn Thr Ala Pro Asn Tyr Pro Ala Tyr Tyr Ser 165 170 175 Asn Ala IleAla Val Ala Ser Thr Asp Gln Asn Asp Asn Lys Ser Ser 180 185 190 Phe SerThr Tyr Gly Ser Val Val Asp Val Ala Ala Pro Gly Ser Trp 195 200 205 IleTyr Ser Thr Tyr Pro Thr Ser Thr Tyr Ala Ser Leu Ser Gly Thr 210 215 220Ser Met Ala Thr Pro His Val Ala Gly Val Ala Gly Leu Leu Ala Ser 225 230235 240 Gln Gly Arg Ser Ala Ser Asn Ile Arg Ala Ala Ile Glu Asn Thr Ala245 250 255 Asp Lys Ile Ser Gly Thr Gly Thr Tyr Trp Ala Lys Gly Arg ValAsn 260 265 270 Ala Tyr Lys Ala Val Gln Tyr 275 10 269 PRT Bacillus 10Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 1015 His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20 2530 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 4045 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 5560 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 65 7075 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 8590 95 Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser ProSer 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr SerArg Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala GlySer Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala ValGly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln TyrGly Ala Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Asn Val Gln SerThr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser MetAla Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln LysAsn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240 Arg Asn His Leu LysAsn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly Ser GlyLeu Val Asn Ala Glu Ala Ala Thr Arg 260 265 11 307 PRT Bacillus 11 MetAsn Gly Glu Ile Arg Leu Ile Pro Tyr Val Thr Asn Glu Gln Ile 1 5 10 15Met Asp Val Asn Glu Leu Pro Glu Gly Ile Lys Val Ile Lys Ala Pro 20 25 30Glu Met Trp Ala Lys Gly Val Lys Gly Lys Asn Ile Lys Val Ala Val 35 40 45Leu Asp Thr Gly Cys Asp Thr Ser His Pro Asp Leu Lys Asn Gln Ile 50 55 60Ile Gly Gly Lys Asn Phe Ser Asp Asp Asp Gly Gly Lys Glu Asp Ala 65 70 7580 Ile Ser Asp Tyr Asn Gly His Gly Thr His Val Ala Gly Thr Ile Ala 85 9095 Ala Asn Asp Ser Asn Gly Gly Ile Ala Gly Val Ala Pro Glu Ala Ser 100105 110 Leu Leu Ile Val Lys Val Leu Gly Gly Glu Asn Gly Ser Gly Gln Tyr115 120 125 Glu Trp Ile Ile Asn Gly Ile Asn Tyr Ala Val Glu Gln Lys ValAsp 130 135 140 Ile Ile Ser Met Ser Leu Gly Gly Pro Ser Asp Val Pro GluLeu Glu 145 150 155 160 Glu Ala Val Lys Asn Ala Val Lys Asn Gly Val LeuVal Val Cys Ala 165 170 175 Ala Gly Asn Glu Gly Asp Gly Asp Glu Arg ThrGlu Glu Leu Ser Tyr 180 185 190 Pro Ala Ala Tyr Asn Glu Val Ile Ala ValGly Ser Val Ser Val Ala 195 200 205 Arg Glu Leu Ser Glu Phe Ser Asn AlaAsn Lys Glu Ile Asp Leu Val 210 215 220 Ala Pro Gly Glu Asn Ile Leu SerThr Leu Pro Asn Lys Lys Tyr Gly 225 230 235 240 Lys Leu Thr Gly Thr SerMet Ala Ala Pro His Val Ser Gly Ala Leu 245 250 255 Ala Leu Ile Lys SerTyr Glu Glu Glu Ser Phe Gln Arg Lys Leu Ser 260 265 270 Glu Ser Glu ValPhe Ala Gln Leu Ile Arg Arg Thr Leu Pro Leu Asp 275 280 285 Ile Ala LysThr Leu Ala Gly Asn Gly Phe Leu Tyr Leu Thr Ala Pro 290 295 300 Asp GluLeu 305 12 281 PRT Bacillus 12 Ser Asp Gly Thr Asp Thr Ser Asp Asn PheGlu Gln Trp Asn Leu Glu 1 5 10 15 Pro Ile Gln Val Lys Gln Ala Trp LysAla Gly Leu Thr Gly Lys Asn 20 25 30 Ile Lys Ile Ala Val Ile Asp Ser GlyIle Ser Pro His Asp Asp Leu 35 40 45 Ser Ile Ala Gly Gly Tyr Ser Ala ValSer Tyr Thr Ser Ser Tyr Lys 50 55 60 Asp Asp Asn Gly His Gly Thr His ValAla Gly Ile Ile Gly Ala Lys 65 70 75 80 His Asn Gly Tyr Gly Ile Asp GlyIle Ala Pro Glu Ala Gln Ile Tyr 85 90 95 Ala Val Lys Ala Leu Asp Gln AsnGly Ser Gly Asp Leu Gln Ser Leu 100 105 110 Leu Gln Gly Ile Asp Trp SerIle Ala Asn Arg Met Asp Ile Val Asn 115 120 125 Met Ser Leu Gly Thr ThrSer Asp Ser Lys Ile Leu His Asp Ala Val 130 135 140 Asn Lys Ala Tyr GluGln Gly Val Leu Leu Val Ala Ala Ser Gly Asn 145 150 155 160 Asp Gly AsnGly Lys Pro Val Asn Tyr Pro Ala Ala Tyr Ser Ser Val 165 170 175 Val AlaVal Ser Ala Thr Asn Glu Lys Asn Gln Leu Ala Ser Phe Ser 180 185 190 ThrThr Gly Asp Glu Val Glu Phe Ser Ala Pro Gly Thr Asn Ile Thr 195 200 205Ser Thr Tyr Leu Asn Gln Tyr Tyr Ala Thr Gly Ser Gly Thr Ser Gln 210 215220 Ala Thr Pro His Ala Ala Ala Met Phe Ala Leu Leu Lys Gln Arg Asp 225230 235 240 Pro Ala Glu Thr Asn Val Gln Leu Arg Glu Glu Met Arg Lys AsnIle 245 250 255 Val Asp Leu Gly Thr Ala Gly Arg Asp Gln Gln Phe Gly TyrGly Leu 260 265 270 Ile Gln Tyr Lys Ala Gln Ala Thr Asp 275 280 13 345PRT Bacillus 13 Leu Arg Gly Leu Glu Gln Ile Ala Gln Tyr Ala Thr Asn AsnAsp Val 1 5 10 15 Leu Tyr Val Thr Pro Lys Pro Glu Tyr Glu Val Leu AsnAsp Val Ala 20 25 30 Arg Gly Ile Val Lys Ala Asp Val Ala Gln Asn Asn PheGly Leu Tyr 35 40 45 Gly Gln Gly Gln Ile Val Ala Val Ala Asp Thr Gly LeuAsp Thr Gly 50 55 60 Arg Asn Asp Ser Ser Met His Glu Ala Phe Arg Gly LysIle Thr Ala 65 70 75 80 Leu Tyr Ala Leu Gly Arg Thr Asn Asn Ala Asn AspPro Asn Gly His 85 90 95 Gly Thr His Val Ala Gly Ser Val Leu Gly Asn AlaThr Asn Lys Gly 100 105 110 Met Ala Pro Gln Ala Asn Leu Val Phe Gln SerIle Met Asp Ser Gly 115 120 125 Gly Gly Leu Gly Gly Leu Pro Ala Asn LeuGln Thr Leu Phe Ser Gln 130 135 140 Ala Tyr Ser Ala Gly Ala Arg Ile HisThr Asn Ser Trp Gly Ala Pro 145 150 155 160 Val Asn Gly Ala Tyr Thr ThrAsp Ser Arg Asn Val Asp Asp Tyr Val 165 170 175 Arg Lys Asn Asp Met ThrIle Leu Phe Ala Ala Gly Asn Glu Gly Pro 180 185 190 Gly Ser Gly Thr IleSer Ala Pro Gly Thr Ala Lys Asn Ala Ile Thr 195 200 205 Val Gly Ala ThrGlu Asn Leu Arg Pro Ser Phe Gly Ser Tyr Ala Asp 210 215 220 Asn Ile AsnHis Val Ala Gln Phe Ser Ser Arg Gly Pro Thr Arg Asp 225 230 235 240 GlyArg Ile Lys Pro Asp Val Met Ala Pro Gly Thr Tyr Ile Leu Ser 245 250 255Ala Arg Ser Ser Leu Ala Pro Asp Ser Ser Phe Trp Ala Asn His Asp 260 265270 Ser Lys Tyr Ala Tyr Met Gly Gly Thr Ser Met Ala Thr Pro Ile Val 275280 285 Ala Gly Asn Val Ala Gln Leu Arg Glu His Phe Val Lys Asn Arg Gly290 295 300 Val Thr Pro Lys Pro Ser Leu Leu Lys Ala Ala Leu Ile Ala GlyAla 305 310 315 320 Ala Asp Val Gly Leu Gly Phe Pro Asn Gly Asn Gln GlyTrp Gly Arg 325 330 335 Val Thr Leu Asp Lys Ser Leu Asn Val 340 345 1419 PRT Artificial Sequence Variation 14 Ala Val Lys Val Leu Gly Ala SerGly Ser Gly Ala Ala Gly Ser Val 1 5 10 15 Ser Ser Ile 15 18 PRTArtificial Sequence Variation 15 Ala Val Lys Val Leu Gly Ala Ser Ser GlyGly Ser Gly Ser Val Ser 1 5 10 15 Ser Ile 16 18 PRT Artificial SequenceVariation 16 Ala Val Lys Val Leu Gly Ala Ala Ser Gly Gly Thr Gly Ser ValSer 1 5 10 15 Ser Ile 17 65 DNA Primers 17 ctatacgcta aagtcctaggggcgrsyrsy rsyrsyrsyr syrsygtcag ctcgattgcc 60 caagg 65 18 21 DNAArtificial Sequence Primer 18 ccctttaacc gcacagcgtt t 21

1. An isolated subtilase enzyme, having improved wash performance in adetergent, as compared to BLSAVI, having an amino acid sequence which isat least 40% identical to the amino acid sequence of the mature BLSAVI,and characterized by that at least one of the active site loops, in saidisolated subtilase, is longer than the corresponding active site loop inBLSAVI, whereby such active site loops regions, in said isolatedsubtilase, is having the minimum amino acid length as specified from thegroup below comprising: (a) the region (both of the end amino acidsincluded) between amino acid residue from 33 to 43 is at least 12 aminoacid long (i.e. at least one amino acid insertion, as compared toBLSAVI); (b) the region (both of the end amino acids included) betweenamino acid residue 95 to 103 is at least 10 amino acids long (i.e. atleast one amino acid insertion, as compared to BLSAVI); (c) the region(both of the end amino acids included) between amino acid residue 125 to132 is at least 9 amino acids long (i.e. at least one amino acidinsertion, as compared to BLSAVI); (d) the region (both of the end aminoacids included) between amino acid residue 153 to 173 is at least 22amino acids long (i.e. at least one amino acid insertion, as compared toBLSAVI); (e) the region (both of the end amino acids included) betweenamino acid residue 181 to 195 is at least 16 amino acids long (ie. atleast one amino acid insertion, as compared to BLSAVI); (f) the region(both of the end amino acids included) between amino acid residue 202 to204 is at least 4 amino acids long (ie. at least one amino acidinsertion, as compared to BLSAVI); and (g) the region (both of the endamino acids included) between amino acid residue 218 to 219 is at least3 amino acids long (i.e. at least one amino acid insertion, as comparedto BlsavI):
 2. The isolated subtilase enzyme of claim 1, wherein saidsubtilase enzyme is a-constructed variant comprising at least oneinsertion of at least one amino acid within at least one of the activesite loops of claim
 1. 3. The isolated subtilase enzyme of claim 2,wherein at least one of said inserted amino acid residue is chosen fromthe group comprising: A, G, S, and T.
 4. The isolated subtilase enzymeof claim 2, wherein at least one of said inserted amino acid residue ischosen from the group of charged amino acid residues comprising: D, E,H, K, and R, more preferably D, E, K and R.
 5. The isolated subtilaseenzyme of claim 2, wherein at least one of said inserted amino acidresidue is chosen from the group of hydrophilic amino acid residuescomprising: C, N, Q, S and T, more preferably N, Q, S and T.
 6. Theisolated subtilase enzyme of claim 2, wherein at least one of saidinserted amino acid residue is chosen from the group of smallhydrophobic amino acid residues comprising: A, G and V.
 7. The isolatedsubtilase enzyme of claim 2, wherein at least one of said inserted aminoacid residue is chosen from the group of large hydrophilic amino acidresidues comprising: F, I, L, M, P, W and Y, more preferably F, I, L, M,and Y.
 8. The isolated subtilase enzyme of claim 2, wherein saidinsertion, in at least one of the active site loops, comprises at leasttwo amino acids, as compared to the corresponding active site loop inBLSAVI.
 9. The isolated subtilase enzyme of claim 2, wherein thesubtilase enzyme comprises at least one insertion selected from thegroup consisting of: G97GASG; G97GM; and G97GAS.
 10. The isolatedsubtilase enzyme of claim 9, comprising at least oneinsertion/modification selected from the group consisting of: 37.03:G97GASG+A98S+S99G+G100A+S101A; 37.06: G97GAA+A98S+S99G+S101T; and 37.04:G97GAS+A98S+S99G.
 11. The subtilase of claim 2, wherein the parentsubtilase is a sub-group I-S1 subtilase.
 12. The subtilase of claim 11,wherein the parent subtilase is chosen from the group comprisingABSS168, BASBPN, BSSDY, and BLSCAR or functional variants thereof havingretained the characteristic of sub-group I-S1.
 13. The subtilase ofclaim 2, wherein parent subtilase, is a sub-group I-S2 subtilase. 14.The subtilase of claim 13, wherein the parent subtilase is chosen fromthe group comprising BLS147, BLS309, BAPB92, TVTHER AND BYSYAB orfunctional variants thereof having retained the characteristic ofsub-group I-S2.
 15. The subtilase enzyme variant of claim 2, furthercomprising at least one further modification.
 16. The subtilase variantof claim 15, wherein said modification is at one or more of thepositions 27, 36, 57, 76, 87, 97,101,104, 120,123, 167,170, 206, 218,222, 224, 235 and
 274. 17. The subtilase variant of claim 16, whereinsaid subtilase belongs to the 1-S2 sub-group and said furthermodification is selected from the group consisting of K27R, *36D, S57P,N76D, S87N, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, Y167, R170,Q206E, N218S, M222A, M222S, T224S, K235L, and T274A.
 18. The variant ofclaim 17, comprising K27R+V104Y+N123S+T274A, N76D+S103A+V104I,N76D+V104A, S87N+S101G+V104N, S101G+V104N, or any other combination ofK27R, N76D, S101G, V104A V104N, V104Y, N123S, and T274A.
 19. Thesubtilase variant of claim 15, wherein said modification is at one ormore of the positions 129, 131,133 and
 194. 20. The variant of claim 19,wherein said subtilase belongs to the I-S2 sub-group and said furthermodification is selected from the group consisting of P129K, P131H,A133D, A133P, and A194P.
 21. The variant of claim 20, wherein saidfurther modification is: Y167A+R170L+P129K, Y167A+R170L+P131H,Y167A+R170L+A133D, Y167A+R170L+A133P, Y167A+R170L+A194P,Y167A+R170N+P129K, Y167A+R170N+P131H, Y167A+R170N+A133D,Y167A+R170N+A133P, Y167A+R170N+A194P, Y167A+R170S+P129K,Y167A+R170S+P131H, Y167A+R170S+A133D, Y167A+R170S+A133P, orY167A+R170S+A194P.
 22. An isolated DNA sequence encoding a subtilase ora subtilase variant of claim
 1. 23. An expression vector comprising anisolated DNA sequence of claim
 22. 24. A microbial host cell transformedwith an expression vector of claim
 23. 25. The microbial host of claim24, which is a bacterium, preferably a Bacillus, especially B. lentus.26. The microbial host of claim 25, which is a fungus or yeast,preferably a filamentous fungus, especially an Aspergillus.
 27. A methodfor producing a subtilase or a subtilase variant, comprising (a)culturing a host of claim 24 under conditions conducive to theexpression and secretion of the subtilase or subtilase variant, and (b)recovering the subtilase or subtilase varian.
 28. A compositioncomprising a subtilase or subtilase variant of claim 1 and a surfactant.29. The composition of claim 28, which additionally comprises anamylase, cellulase, cutinase, lipase, oxidoreductase, or anotherprotease.
 30. Use of a subtilase or a subtilase variant of claim 1 or anenzyme composition of claim 28 in a laundry and/or a dishwash detergent.